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Full text of "Subunit-specific determinants of function and pharmacology of nicotinic acetylcholine receptors"

SUBUNIT-SPEC TT ^IC DETERMINANTS OF 
FUNCTION AND PHARMACOi OGY OF NICOTINIC ACETYLCHOLINE 

RECEPTORS 






BY 
MICHAEL M. FRANCIS 









A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1998 



THIS DISSERTATION IS DEDICATED TO MY PARENTS 


















ACKNOWLEDGMENTS 

I look back on the last five years and wonder why it took me so long to accomplish the 
things which I have accomplished. Upon reflection, I realize how much longer it would 
have taken without the patience, understanding and guidance of family, friends and 
instructors. I would like to thank my father, Miles, mother, Sandy, and sister, Kathy, for 
putting up with five years of having to take a backseat to my pursuit of this degree. It has 
been a real benefit to me to know that I have a wonderful family to fall back on whenever I 
have needed to. It has been a great privilege to have the unquestioning support of my 
family especially in light of the fact that they have no clue as to exactly what I am studying 
or why it is taking so long. 

I would also like to thank the many friends, old and new, near and far, who have helped 
encourage, distract, intoxicate and otherwise lend a vitality to my life which really keeps 
graduate school in perspective. In particular, I would like to thank the many friends I have 
made in Gainesville during the course of graduate school for not allowing me to waste the 
few free hours I have on worrying about what I could be doing in the lab. 

I would like to thank the members of my graduate committee past and present, Dr. Peter 
Anderson, Dr. Jeff Harrison, Dr. Ben Horenstein, Dr. Mike King, Dr. Bob Lenox, and 
Dr. Janet Zengel, for tneir time and effort in helping me to achieve my goals as a scientist 
and as a person. I would like to thank the current members of the Papke laboratory (Nik, 
Gill, Anatolii, Julia and Chris) as well as past members (Clare, Hugo, Amy, Rick, Becky 
and Wayne) for their encouragement and intellectual as well as technical support. 

Finally, I would like to express my deep gratitude to my mentor, Dr. Roger Papke, 
foremost for his guidance throughout my course of study and also for his patience in 



in 



understanding the times when I needed a break from my course of study. The free 
exchange of ideas with him has been indispensable in transforming a psychology major 
with a vague notion of wanting to study synaptic transmission into a successful 
neuroscientist. 












IV 



TABLE OF CONTENTS 

page 

ACKNOWLEDGMENTS iii 

ABSTRACT vii 

CHAPTERS 

1. INTRODUCTION 1 

Nicotinic Receptor Gene Family 3 

General Features of Muscle-type Receptors 3 

Cloning of the Neuronal nAChRs 4 

General Features of Neuronal nAChRs 5 

Function of Nicotinic Acetylcholine Receptors 6 

Pharmacology and Electrophysiology in the Study of nAChR 6 

Neuromuscular Junction nAChR 12 

In vivo Subunit Composition and Distribution of Neuronal nAChRs 15 

Functional Roles of Neuronal nAChRs 19 

Sensitivity to Noncompetitive Inhibitors 28 

Structure of nAChRs 33 

General Structural Features 34 

N-Terminal Domain and Agonist Binding Site 37 

Transmembrane Domains 1-4 (TM1-4) and Cytoplasmic Loop 39 

Relating Structure to Function for Neuronal nAChRs 46 

2. MATERIALS AND METHODS 48 

Chemicals and Synthesis 48 

Production of Chimeras and Sequencing 49 

Preparation of RNAs and Oocyte Expression 49 

Electrophysiology 50 

Two-Electrode Voltage Clamp 50 

Cut-Open Vaseline Gap Voltage Clamp 51 

Experimental Protocols and Data Analysis 52 

3. RESULTS 56 

Structural Determinants of Sensitivity to the TMP Family of NCIs 56 

Muscle Delta Subunit Effects 56 

Neuronal Beta Subunit Effects 73 

Mechanism of Inhibition of nAChRs by bis-TMP-10 87 

Inhibition by Bis-TMP-10 Is Independent of Voltage 87 

Inhibition of nAChRs by QX-314 97 



Requirements for Long-Term Inhibition by Bis-TMP-10 105 

Inhibition by an Amphipathic Analogue of Bis-TMP-10 116 

Inhibition by ATMP-10 Independent of Activation by Agonist 119 

Voltage-Dependence of Inhibition by ATMP-10 121 

TMP Protects aipi(p4TM2)y8 Receptors from Inhibition by ATMP-10 126 

4. DISCUSSION 127 

The Mechanism of Action of Mecamylamine Is Distinct from that of Bis-TMP-10.... 128 

TM2 Determines Kinetics of Long-Term Inhibition by Bis-TMP-10 129 

Significance of Voltage-Independent Inhibition 131 

Inhibition by Bis-TMP-10 Is Distinct from Inhibition by QX-314 133 

Bis-TMP-10 and QX-314 Do Not Compete for the Same Site 134 

Significance of Compound Length Requirement for Long-Term Inhibition 137 

5. SUMMARY AND CONCLUSIONS 142 

REFERENCES 147 

BIOGRAPHICAL SKETCH 161 















VI 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfillment of the 

Requirements for the Degree of Doctor of Philosophy 

SUB UNIT-SPECIFIC DETERMINANTS OF 
FUNCTION AND PHARMACOLOGY OF NICOTINIC ACETYLCHOLINE 

RECEPTORS 

By 

Michael M. Francis 
December, 1998 



Chairman: Roger L. Papke 
Major Department: Neuroscience 



Some noncompetitive inhibitors (e.g., ganglionic blockers) exhibit selectivity for the 
inhibition of neuronal nicotinic acetylcholine receptors (nAChRs). The main goal of the 
present study is to characterize the mechanism of selective long-term inhibition of neuronal 
and muscle-neuronal chimeric nAChRs by bis-TMP-10 (bis (2,2,6,6-tetramethyl-4- 
piperidinyl) sebacate or BTMPS), a bifunctional form of the potent ganglionic blocker 
tetramethylpiperidine. Long-term inhibition of neuronal nAChRs by bis-TMP-10 has been 
previously demonstrated to arise, at least in part, from the binding of the bis- compound to 
neuronal beta subunits. In this study, long-term inhibition is demonstrated to be dependent 
upon the presence of sequence element(s) within the pore-lining second transmembrane 
domain (TM2) of either neuronal beta subunits or muscle delta subunits; however, for 
either class of subunit, because the onset of inhibition does not appear to be strongly 
voltage-dependent, the inhibitor binding site itself does not appear to be contained within 
the segment of the channel pore influenced by the membrane electric field. In the case of 
the neuronal beta subunits, long-term inhibition is also not affected by preapplication of the 



Vll 









open-channel blocker QX-314. Furthermore, we demonstrate a compound length 
requirement for long-term inhibition which would be consistent with binding to multiple 
sites contributed by separate subunits (either beta or delta) located on the extracellular 
portion of the receptor. Our results may imply that bis-TMP-10 interacts with an 
activation-sensitive element, the availability of which may be regulated by sequence in the 
TM2 domain. It is interesting to note that the mechanism of inhibition by bis-TMP-10 
appears to be distinct from that of other ganglionic blockers such as mecamylamine. 
Knowledge of the mechanism underlying the basis for inhibition by pure antagonists may 
be useful for consideration of observations of mixed agonist/antagonist properties of certain 
experimental therapeutics for nicotinic receptors. Furthermore, if bis-TMP-10 is binding to 
an activation-sensitive element distinct from TM2, it may be possible to use sensitivity to 
inhibition by this compound to investigate the structural changes which take place with 
channel gating. 



Vlll 



CHAPTER 1 
INTRODUCTION 



The idea that osmosis and secretion across a biological membrane could be regulated by 
pores in the membrane dates back to the work of Ernst Brucke in the mid- 19th century 
(Brucke, 1843). Only much more recently has the critical role of membrane pores in 
nervous system function come to be recognized. With the almost simultaneous description 
of the ionic basis of action potential generation and propagation by Alan Hodgkin and 
Andrew Huxley (1952) and the observation of quantal changes in electrical potential (called 
miniature endplate potentials or MEPPs) during voltage recordings at the endplate of the 
neuromuscular junction by Paul Fatt and Bernard Katz (1951), the electrochemical nature 
of communication between cells of the nervous system (neurons) began to emerge. 
However, it was only much later still that changes in membrane permeability to ions could 
be definitively linked to the opening and closing of membrane pores or ion channels as they 
would come to be called. Katz and Miledi ( 1972) were able to demonstrate that the 
electrical noise in their recordings increased with application of the endogenous activator of 
muscle fibers, acetylcholine, to the muscle fiber. Via noise analysis, these investigators 
were able to infer that this noise increase could be correlated with opening of individual ion 
channels in the post-synaptic membrane. Their analysis even went so far as to predict an 
estimate for average open time (length of time a channel is conducting before closing) and 
conductance (capacity of a channel to allow ions to permeate) of individual channels. The 
subsequent refinement of techniques such as the voltage-clamp (Anderson and Stevens, 
1973) together with the development and application of improved techniques such as the 
patch clamp (Neher and Sakmann, 1976) allowed direct measurement of the opening and 
closing of ion channels. More recent studies employing these improvements have, in 



general, confirmed much of the theory suggested by the work of earlier 
electrophysiologists. Furthermore, the early studies by investigators such as Hodgkin. 
Huxley and Katz have provided the framework for consideration and interpretation of 
research even today. 

The process of neuronal communication is now commonly referred to as synaptic 
transmission and, in general, consists of the release and subsequent diffusion of a chemical 
messenger or neurotransmitter across junctions between neurons called synapses. Upon 
reaching the post-synaptic neuron, neurotransmitter binds to post-synaptic sites (receptors) 
coupled to closed pores in the membrane. Binding of the neurotransmitter to these 
receptors triggers a change in the structure of the pore so that ions can flow freely through 
the ligand-gated ion channel, so called because the binding of a ligand opens the ion 
channel pore. The resulting redistribution of charged ions causes a change in the electrical 
potential across the membrane and hence, elicits a response in the post-synaptic cell. This 
response can be either inhibitory or excitatory depending upon the particular type of 
receptors present on the post-synaptic cell. If an excitatory response reaches a certain 
threshold, it will cause an action potential to be generated and propagated by the activity of 
another class of ion channels, voltage-gated sodium channels (a change in the distribution 
of charge opens the ion channel pore). When the action potential reaches the presynaptic 
terminal, calcium enters via the activation of voltage-gated calcium channels. This calcium 
influx initiates the events involved in the release of packets containing neurotransmitter 
(synaptic vesicles) and, for excitatory neurotransmitters, the process repeats itself. 
Although the efficacy of this process can be modulated on a cellular and even subcellular 
level, this general form of electrochemical coupling underlies all rapid transmission in the 
nervous system. 

Since the time of these early studies, the ongoing development of biochemical and 
molecular biological techniques has provided for the identification of a great diversity of ion 
channels in the nervous system and permitted the study of homogeneous populations of ion 



channel subtypes in isolated systems which allow more detailed analyses of the relationship 
between ion channel structure and function. The particular subtype of ion channel found at 
the neuromuscular junction, the nicotinic acetylcholine receptor (nAChR), remains a 
prototype system for these studies. Additionally, cloning of the neuromuscular junction 
nAChR has permitted the identification of a great diversity of nAChR subtypes in the 
central and peripheral nervous system. 

Nicotinic Receptor Gene Family 

The genes encoding nicotinic acetylcholine receptors are members of a superfamily of 
genes coding for ligand-gated ion channels which share considerable sequence homology 
and seem to have roughly equivalent membrane topographies. Other receptors encoded for 
by members of the gene family include GABA (gamma-amino butyric acid), glycine and 
5HT3 (serotonin) receptors. All of the proteins encoded by members of this gene family 
share a characteristic 13 residue loop between disulfide-linked cysteine residues in the N- 
terminal domain (Kao and Karlin, 1986). Additionally, although each of these receptor 
types differs in selectivity for agonist and relative ionic permeabilities, they share certain 
essential functional properties. Specifically, agonist binds to distinct sites on the receptor 
complex causing a conformational change in the protein which allows ions (principally 
sodium, calcium and potassium in the case of nAChR and 5HT3 receptors or chloride in 
the case of GABA and glycine receptors) to flow down electrical and chemical gradients in 
relative proportions specific to each receptor type. 

General Features of Muscle-type Receptors 

Muscle-type nAChRs are the best characterized of the ligand-gated ion channels and as 
such are a model system for the study of structure-function relationships in the gene family. 
Muscle-type nAChRs are highly concentrated in the junctional folds at the endplate of the 
neuromuscular junction where they serve to transmit neuronal impulses to the muscle fiber. 



Much of the initial characterization of the structural features of muscle-type nAChR can be 
attributed to the ready availability of large quantities of a homologue of muscle-type nAChR 
in the electric organs of the Torpedo ray. From biochemical experiments on Torpedo 
nAChR, it was possible to deduce a putative membrane topology and structural 
organization which has proven to be, for the most part, conserved in mammalian muscle- 
type nAChR. 

Muscle-type nAChRs are pentameric complexes, consisting of four distinct protein 
subunits (alply5) with protein molecular weights of about 50,000 (alphal), 53,700 
(betal), 56,300 (gamma), and 57,600 (delta) in the ratio of 2:1:1:1 (Conti-Tronconi etai, 
1982). Two molecules of acetylcholine bind to sites believed to be located at the interface 
of the alpha subunits with the delta and gamma subunits (Blount and Merlie, 1989; 
Pedersen and Cohen, 1990; Sine and Claudio, 1991) to activate the receptor. It has now 
become apparent that the function of muscle-type nAChRs is developmentally regulated via 
the substitution of the epsilon subunit for the gamma subunit in the adult form of the 
receptor (Mishina et al, 1986). 

Cloning of the Neuronal nAChRs 

N-terminal sequencing of the muscle nAChR alphal subunit (Raftery etai, 1980) and 
production of degenerate oligonucleotides allowed for the identification of a cDNA 
encoding the muscle alpha subunit (Noda et al, 1982) and subsequent cloning of cDNAs 
encoding the muscle beta, gamma and delta subunits (Noda et al, 1983a, 1983b). 
Subsequently, a neuronal homologue of alphal was cloned via low stringency 
hybridization screening of a PC 12 cell cDNA library with an oligonucleotide designed from 
the muscle alphal sequence (Boulter et al, 1986). At about the same time, another group 
working independently cloned a second neuronal alphal homologue from a chick brain 
cDNA library (Nef et al, 1988). Based on the conservation of a set of vicinal cysteines 
additional to the cysteine pair characteristic to all members of the gene family and shown to 



be important for formation of the agonist binding sites (Kao and Karlin, 1986), these 
neuronal homologues were eventually designated as alpha3 and alpha2 respectively. 
Subsequent identification of additional neuronal homologues of the muscle alpha subunit 
clone from both rat and chick proceeded rather quickly after the identification of alpha2 and 
alpha3 (Boulter et al, 1990; Couturier et al, 1990a, 1990b; Deneris et al., 1988; Goldman 
etal., 1987; Schoepfer era/., 1988; Seguela etal, 1993; Wada etal, 1988). All neuronal 
subunits which share the conserved vicinal cysteine residues in the N-terminal domain have 
been designated as alpha subunits while the first non-alpha subunit was designated beta 2 
based on it's ability to substitute for the betal subunit of muscle nAChR. Subsequently, 
certain other non-alpha subunits have been designated as neuronal beta subunits because of 
their ability to form functional receptors when expressed in pairwise combination with 
neuronal alphas. 

General Features of Neuronal nAChRs 

Eight neuronal nicotinic receptor subunits (alphas2-9) and three beta (betas2-4) subunits 
have been cloned to date (for review see (Lindstrom, 1996)). The receptors formed from 
these subunits fall into two major groups. Based on heterologous expression studies of 
alphas2-4, it has been demonstrated that these alpha subunits require coexpression with 
either beta2 or beta4 for function and form heteromeric receptors consisting of nonidentical 
subunits. Data from studies of heterologously expressed receptors indicate that both the 
alpha and beta subunits influence activation and inhibition characteristics of neuronal 
nAChRs (Luetje and Patrick, 1991). Based on these data and by analogy with the situation 
for muscle-type receptors, it is hypothesized that the agonist binding sites of neuronal 
nAChR lie at the interface of the alpha subunits with the beta subunits. The alpha5 subunit 
appears to function as more of a structural or modulatory subunit although functional 
effects of this subunit have recently been described (Wang et al., 1996). While the alpha5 
protein does contain the conserved vicinal cysteine residues characteristic of alpha subunits, 



it lacks other conserved residues in the N-terminal domain which are believed to contribute 
to the ACh binding site (see below). Alpha6 appears to require coexpression with the beta4 
subunit in particular for function (Gerzanich et ai, 1996). A functional role for the beta3 
subunit in heterologous expression systems has not been described thus far although recent 
evidence suggests that it is incorporated into heteromeric receptors in the brain (Forsayeth 
and Kobrin, 1996). 

The second major category of neuronal nAChR subtypes is defined by subunits which 
have the ability to function as homomeric receptors (receptors formed entirely from 
identical subunits) and is comprised of alphas7-9. Alpha8 has only been found in chick 
thus far where it most likely functions both as a homomer and as a heteromer with alpha7 
(Gerzanich et al, 1993). In addition to possessing unique and heterogeneous functional 
properties (see below), these receptors exhibit unique pharmacological characteristics. 

Function of Nicotinic Acetylcholine Receptors 

Pharmacology and Electrophysiology in the Study of nAChRs 

Before the development of molecular biology, ion channels were generally categorized 
according to their pharmacology. For example, although the endogenous agonist for the 
nicotinic acetylcholine receptor is acetylcholine, the receptor can be distinguished 
pharmacologically on the basis of high affinity binding of the drug nicotine and other 
agonists (e.g., cytisine). This property distinguishes the nicotinic receptor from another 
classification of acetylcholine receptors in the nervous system which bind muscarine with 
relatively high affinity, the muscarinic receptors. Muscarinic acetylcholine receptors are not 
directly coupled to ion channels but instead serve to, among other things, modulate 
neuronal excitability via initiation of second messenger cascades through the action of a 
class of proteins known as GTP-binding proteins or simply G-proteins (Hille, 1992). This 
class of acetylcholine receptors will not be discussed further here. 



Although the identification of new ion channel subtypes currently relies, for the most 
part, on molecular biological rather than pharmacological techniques, pharmacology still 
provides a strong tool to identify the distribution and study functional relationships of 
particular ion channel subtypes. Pharmacological agents are generally described as either 
agonists or antagonists. Agonists bind to the receptor at a particular site and activate the 
receptor. For example, the neurotransmitter acetylcholine is an agonist of the nicotinic 
acetylcholine receptor. In contrast, antagonists act to inhibit channel function via a variety 
of different mechanisms (discussed below). The relative ability of particular agonists and 
antagonists to either activate or inhibit channel function also provides a tool to distinguish 
between different ion channel subtypes. In addition to their role as channel activators and 
inhibitors, agonists and antagonists, particularly high affinity agonists and antagonists, can 
be valuable tools in binding experiments examining the tissue distribution of particular ion 
channel subtypes. 

Although a great deal can be learned by studying the binding characteristics of ligands to 
specific receptor sites, the study of ion channel function requires a technique whereby the 
functional effects of ligand binding can be observed. The direction of flow of ions is 
determined by the relative ionic concentrations present on the interior and exterior of the 
cell. Because the cell membrane acts as a barrier to the free diffusion of ions between the 
cytoplasm and the extracellular space, ATP-activated ion transport systems can bring about 
the production of ionic gradients which accumulate with respect to the interior and exterior 
of the cell. Potassium ions are concentrated intracellularly while sodium ions are 
concentrated extracellularly. This gradient is actively maintained by the activity of a Na-K 
pump which exchanges intracellular sodium for extracellular potassium. Because this 
gradient represents potential energy it is referred to as the membrane potential. Because the 
membrane potential arises from the separation of charged ions, it necessarily has both an 
electrical and chemical component. When the membrane permeability for a specific ion 
increases (by receptor activation, for example) it is possible to describe an electrical 



8 

potential which exactly counterbalances the chemical potential arising from the 
concentration gradient. At this potential, there will be no flow of ions across the 
membrane. This potential is known as the ionic equilibrium potential. In contrast, the 
magnitude and direction of net charge flow through a nonspecific cation channel like 
nAChR is dictated by the voltage difference between the membrane potential (Vm) and the 
combined and weighted equilibrium potentials for each permeant ion (the reversal potential 
or Erev), a quantity known as the driving force (Vm-Erev). 

Electrophysiology takes advantage of the fact that we can treat the flow of ions across a 
membrane in a similar manner to the flow of electrons in electricity. That is, the movement 
of charge arising from the activation of ion channels can be described by Ohm's law: 

I=VG 
where 
I=current 

V=voltage or driving force 
G=conductance (the inverse of resistance) 

In other words, the amount of current (movement of charge) observed involves both the 
magnitude of the electrical potential gradient and the ability of current to flow through the 
conductor (in this case, the ion channels). Many electrophysiology experiments are carried 
out under conditions of voltage clamp where the membrane potential is held constant. In 
this case, it is possible to relate the currents observed directly to the conductance of the 
membrane (or more specifically, the conductance of the ion channels in the membrane). 
For a system such as a heterologous expression system (e.g., Xenopus oocyte), in which 
the expressed channels are of a homogeneous nature, a constant number of channels of 
equivalent conductance are present. Therefore, it is useful to expand Ohm's law to: 



I=VNp Y 
where 

N=# of ion channels 

p =probability of the channels to be open 
and y=the conductance of a single channel 

From the discussion above, it is apparent that the electrical potential gradient is defined by 
the driving force on the ions (Vm-Erev). Because in an oocyte voltage-clamp experiment, 
Vm-Erev, N and y are all constant, it is possible to directly relate current to the probability 
of the channels to be open. For a ligand-gated channel, open probability depends upon 
agonist concentration. Thus, it is possible to relate directly agonist concentration to 
receptor response. The ability to reliably quantify the activation and inhibition of specific 
receptor subtypes in response to the application of specific drugs has allowed a detailed 
description of the mechanism of action of a variety of different drugs and, furthermore, has 
permitted the comparison of functional differences across receptor subtypes. For example, 
although a binding experiment may indicate that a particular agonist has a similar affinity 
across receptor subtypes, electrophysiological characterization of the effects of this drug 
may reveal a selectivity for the activation of a particular subtype. In the case of neuronal 
nAChRs, the agonist cytisine exhibits a high binding affinity for beta2 -containing 
receptors; however, voltage clamp studies of heterologously expressed receptors indicate 
cytisine to be only a partial agonist with very low efficacy for receptors containing the beta2 
subunit and a full agonist for receptors containing the beta4 subunit (Papke and 
Heinemann, 1994). Similarly, the agonist DMPP is more efficacious on receptors 
containing the beta2 subunit and less so on receptors containing the beta4 subunit (Luetje 
and Patrick, 1991). Thus, binding affinity can not be viewed as a reliable indicator of 
agonist efficacy or potency. By using electrophysiology in combination with molecular 



10 

biological approaches it is possible to localize the structural elements specific to individual 
subunits which underlie each of these characteristics. 

The voltage-clamp technique has permitted detailed analysis of the mechanism of action 
of inhibitors of ion channel function as well. The mechanism of antagonist action is varied 
and can be quite complex; however, antagonists can be broadly classified as either 
competitive or noncompetitive. Competitive antagonists bind to the same site as agonist but 
do not activate the receptor and therefore compete with agonist for occupation of the 
receptor binding site. A hallmark of this type of antagonist activity is the observation that 
high concentrations of agonist are able to compete off more moderate concentrations of the 
competitive antagonist and fully activate a population of channels, albeit at a higher 
concentration than if the antagonist was not present at all. In pharmacological terms, the 
presence of the competitive antagonist would shift the concentration-response curve for the 
agonist to the right indicating an apparent change in potency while no change in efficacy or 
maximal response would be observed. As is the case for agonists, certain antagonists 
show selectivity for the inhibition of particular receptor subtypes. For example, in the case 
of neuronal nAChRs, beta2-containing receptors exhibit more prolonged inhibition by the 
competitive antagonist neuronal bungarotoxin compared with beta4-containing receptors. 
Structural elements underlying this difference in sensitivity have been localized to the N- 
terminal 121 amino acids of the beta subunit (Papke etal., 1993). In addition, high affinity 
competitive antagonists (e.g., ot-bungarotoxin), like potent agonists, can be extremely 
valuable as reagents for binding experiments examining the tissue distribution of particular 
receptor subtypes. 

Noncompetitive antagonists act at sites that are distinct from the agonist binding sites 
and are, in some cases, also use-dependent meaning that they require the presence of 
agonist in order to inhibit the receptor. The study of the mechanism of action of 
noncompetitive inhibitors has proven to be quite complex and has provided a great deal of 
information about ion channel function. However, in general the activity of noncompetitive 



11 

inhibitors is characterized by a reduction in the efficacy of agonist for activation of the 
receptor. Thus, in contrast to the situation for competitive inhibitors in which high 
concentrations of agonist can alleviate the effects of the inhibitor, the effects of 
noncompetitive inhibitors remain unaffected by application of high concentrations of 
agonist. It should be noted that, in the case of use-dependent noncompetitive inhibitors, 
which require prior activation of the channel, a low agonist concentration can affect the 
amount of inhibition observed for a population of channels because only channels which 
have been activated are available for inhibition. 

The most well described class of noncompetitive inhibitors are known as open-channel 
blockers (Neher and Steinbach, 1978). Open-channel blockers are generally small, 
charged molecules which act by occupying a site within the ion channel pore and physically 
occluding ion permeation. Because open channel blockers do bind within the membrane- 
spanning region of the ion channel, their binding and unbinding can be affected by the 
membrane electric field, an electric field created by the voltage gradient across the 
membrane. By analyzing the degree to which inhibition is affected by membrane potential, 
it is possible to use this feature of open-channel block to assess the depth of an inhibitor 
binding site within the channel pore. Accordingly, open-channel blockers have been 
extremely useful in the process of identifying structural components of the receptor which 
contribute to the pore-lining region. A second more heterogeneous and less well 
characterized class of noncompetitive inhibitors are known as allosteric inhibitors because 
they are not believed to physically occlude ion channel permeation but most likely bind to a 
site outside the pore-lining region and act to stabilize the receptor in a closed configuration 
(Papke and Oswald, 1989). In general, inhibition by this class of compounds is not state- 
dependent (i.e., does not require prior activation of the channel). Additionally, there is 
some evidence for a class of inhibitors with characteristics intermediate to those of open- 
channel blockers and allosteric inhibitors (Papke etai, 1994). For this class of 



12 

compounds, inhibition seems to require prior activation of the channel but does not appear 
to have an appreciable voltage-dependence. 

Channel block by agonist constitutes a final type of noncompetitive inhibition of 
nAChR. It has been well documented in the literature that, at high concentrations, 
acetylcholine and a number of acetylcholine analogues can have secondary inhibitory 
effects on musc\e-type/Torpedo nAChRs (Arias, 1996; Marshall etai, 1991; Ogden and 
Colquhoun, 1985; Sine and Steinbach, 1984; Tonner etai, 1992). Data from this 
laboratory suggest that nicotine can have a similar effect on certain subtypes on neuronal 
nAChR (de Fiebre et al, 1995). In particular, the oc3p2a5 subtype of neuronal nAChR 
seems to exhibit enhanced sensitivity to secondary inhibition by both ACh and nicotine 
(Francis et al, unpublished observations). Moreover, many of the experimental new 
nicotinic agents being considered for clinical development are in fact only partial agonists 
for high affinity nicotinic receptors and also share, with nicotine, the ability to function as 
antagonists. The identification and characterization of subtype-specific structural 
determinants of pharmacological sensitivity to each of the classes of drugs described above 
remains of major importance as it could prove critical in developing more effective and 
more specific pharmacological agents for nAChRs. 

Neuromuscular Junction nAChR 

As a principal component of the most well studied synapse in neurophysiology, the 
muscle-type nAChR is the prototype ion channel against which all others are compared. 
However, in many respects, the neuromuscular junction is one of the most specialized 
synapses in the body. The major function of muscle nAChR seems to be the faithful 
transmission of pre-synaptic impulses to the post-synaptic muscle fiber and the 
organization of the synapse serves to optimize nAChR activation for this role. At the 
mature neuromuscular junction, the surface of the muscle fiber is characterized by a series 
of invaginations. Nicotinic AChRs are concentrated at the tips of these folds (at a density 






13 

of about 20, 000 binding sites per urn 2 ) in a location known as the endplate. The 
presynaptic terminals of the nerve contact the muscle fiber in this endplate region, bringing 
the release sites for neurotransmitter (the active zone) and the post-synaptic receptors 
together within a distance of about 500 A at the synaptic cleft. When an action potential 
causes release of synaptic vesicles containing ACh, the post-synaptic receptors are rapidly 
activated initiating a local change in the membrane potential of the muscle fiber. This 
change in membrane potential activates voltage-gated sodium channels located deep within 
the junctional folds and a muscle action potential can be generated. A safety factor of about 
10 in terms of excess acetylcholine and receptors is built in to this synapse in order to 
ensure faithful signal transmission. Furthermore, the presence of acetylcholine esterase, an 
enzyme which cleaves acetylcholine, in the extracellular matrix of the synaptic cleft ensures 
that ACh is present for only a very short time. Thus, each ACh receptor probably only 
binds ACh once during a single synaptic event and the termination of the response is 
mediated by the intrinsic rate of deactivation or closing of the receptor (Katz and Miledi, 
1973). This organization permits rapid and efficient signaling of even high frequency 
impulses between the nerve and the muscle fiber which it contacts. 
Functional States of Muscle nAChR 

Exploration of the role of nAChR in mediating transmission at the nerve-muscle synapse 
has led to an understanding of the gating transitions associated with ion channel function 
itself. After studying the response properties of muscle-type nAChR to acetylcholine in the 
presence of a variety of choline derivatives and the esterase inhibitor prostigmine, del 
Castillo and Katz (1957) were able to conclude that "the first step in a depolarizing end- 
plate reaction is the formation of an intermediate, inactive compound between drug and 
receptor" (p. 369). This result eventually led to a kinetic model for the functional 
transitions involved in the activation of nAChR. Although subsequent models have grown 
increasingly complex, the most basic kinetic model consists of three general states of the 
receptor: an unoccupied closed state, an agonist bound closed state and the open state. 



14 

Subsequent analyses have demonstrated that, at high agonist concentrations, activation is 
predominantly associated with the binding of two molecules of acetylcholine and that the 
prolonged presence of agonist at the receptor can induce a non-conducting state which has a 
high affinity for ligand, known as a desensitized state. Thus, a reasonably accurate 
description of most response properties of nAChR can be given by the relation: 

R ; » AR< » A 2 R< *A 2 R*; *• RD 

where 

A=agonist 

R=receptor 

A2R*=agonist bound open state 

D= desensitized state 

The arrows represent transitions between discrete states which proceed at characteristic 
rates. The direct measurement of rates for transitions between states was made possible by 
the development of the patch clamp technique (Hamill et al, 198 1 ; Neher and Sakmann, 
1976). During the analysis of single channel responses to the agonist suberyldicholine, 
Colquhoun and Sakmann (1981) observed what appeared to be short, non-conducting 
states within the current responses of single channels. Analysis of these gaps or fast 
closures led Colquhoun and Sakmann to conclude that the response to a single agonist 
binding event consists of a burst of activity. A burst is represented by transitions between 
the agonist bound closed state and agonist bound open state while longer closed states are 
predominantly associated with extraburst closures and the dissociation of agonist. Other 
long closed states which may observed include desensitized states and blocked states. 






15 



Physiological Characteristics of Muscle versus Neuronal nAChRs 

Although neuronal and muscle nAChR exhibit many similar functional characteristics, 
there do exist significant differences. In particular, neuronal nAChR subtypes generally 
have a higher calcium permeability than muscle nAChR (Mulle et ai, 1992; Rogers and 
Dani, 1995; Vernino etal, 1992, 1994). Regulation of calcium influx via neuronal 
nAChRs could activate second messenger pathways which play a role in processes as 
diverse as neuronal survival, transmitter release and synaptic strengthening. For example, 
calcium influx via nAChRs on neurons of the medial habenula has been shown to be 
sufficient to activate a calcium-dependent chloride conductance and, in addition, cause a 
decrease in the response of GABAa receptors (Mulle et ai, 1992). The alpha7 subtype of 
neuronal nAChR in particular has been demonstrated to have a very high calcium 
permeability and much interest has focused on potential functional roles for this receptor 
subtype. In addition to differences in calcium permeability, neuronal and muscle nAChRs 
differ in their capacity to conduct outward current at positive potentials. Muscle nAChRs 
exhibit a linear current- voltage relation (equal amount of outward current at positive 
potentials as inward current at negative potentials) while neuronal nAChRs exhibit an 
inwardly rectifying current-voltage relationship (greater inward current at negative 
potentials than outward current at positive potentials). Recent evidence indicates that, 
similar to voltage-gated potassium channels and glutamate receptors, the inward 
rectification of neuronal nAChRs is mediated via channel block by intracellular polyamines 
at positive potentials (Haghighi and Cooper, 1998). 

In vivo Subunit Composition and Distribution of Neuronal nAChRs 

As is apparent from the number of genes encoding subunits for neuronal nAChRs, there 
is a much greater diversity of neuronal nAChR in terms of subtype, localization and 
presumed functional role than is the case for muscle-type nAChR. Each of the various 
neuronal nAChR subunits appears to have a tissue-specific distribution (Wada etal, 1989) 



16 

and there is potential for a great variety of subunit combinations to appear in the nervous 
system. Functional nicotinic receptors are present both centrally in the brain and spinal 
cord and peripherally on neurons of the autonomic ganglia. In general, the typical subunit 
composition of neuronal nAChR from peripheral ganglia appears to be distinct from the 
most widely expressed subunit combinations centrally. However, evidence from a number 
of studies supports the notion that a variety of subunit combinations exist both centrally and 
peripherally. 
Peripheral Neuronal nAChRs 

It is apparent that receptors containing the alpha3 and beta4 subunits are the predominant 
nAChR of the peripheral ganglia. However, it remains unclear to what extent these 
subunits coassemble with other nicotinic subunits and the description of receptor subunit 
composition on individual neurons remains problematic. In fact, it appears that multiple 
nAChR subtypes can exist on the same ganglionic neuron (Conroy and Berg, 1995; Poth et 
ai, 1997; Vernallis et ai, 1993). Furthermore, in the case of chick ciliary ganglion 
neurons, it has been shown that the alpha3 subunit nearly always coassembles with the 
beta4 subunit while the alpha5 subunit always coassociates with the alpha3 and beta4 gene 
products. About 20% of these synaptic ciliary ganglion nAChR also contain the beta2 
subunit (Conroy and Berg, 1995). To add further complexity, an additional subtype of 
nAChR containing the alpha7 gene product is present but seems to exhibit a non-synaptic 
or perisynaptic distribution on the neuron (Vernallis et ai, 1993; Wilson Horch and 
Sargent, 1995). These findings are discussed in more detail below. 
Central Neuronal nAChRs 

It has been well established that the nAChR of the neuromuscular junction can be bound 
nearly irreversibly by the snake venom toxin a-bungarotoxin (a-BTX) and that this ligand 
labels putative receptor sites in brain. However, studies employing labeling of central 
nAChR by agonists such as acetylcholine or nicotine demonstrate a labeling pattern quite 
distinct from that of a-BTX (Clarke et ai, 1985) and, in addition, early studies using 



17 

heterologously expressed heteromeric neuronal nAChR show these receptors to be 
insensitive to inhibition by a-BTX (Deneris et al, 1988). With the cloning of the alpha7 
gene product (Couturier et al, 1990; Schoepfer et al, 1990; Seguela et al, 1993), it 
became apparent that a distinct population of nAChR with relatively low binding affinity for 
the traditional nicotinic agonists and very high affinity for a-BTX exist in brain. 
Therefore, it is useful to divide the description of central neuronal nAChR subtypes into 
two general populations: those that bind nicotinic agonists with high affinity and are 
insensitive to inhibition by a-BTX and those nAChR which bind agonist with relatively 
low affinity and are sensitive to low concentrations of a-BTX. 
High Affinity Brain nAChRs 

Towards describing the high affinity brain nAChR population, Whiting and Lindstrom 
(1986) demonstrated that an antibody raised to chick neuronal nAChR (mAb 270) also 
cross-reacts with rat neuronal nAChR and is able to bind greater than 90% of the high 
affinity nicotine binding sites in detergent extracts of rat brain. It has since been shown that 
mAb 270 binds what is now designated the beta2 subunit and this antibody has been used 
to show the distribution of beta2 subunits in rat brain (Swanson et al, 1987). Via analysis 
of immunoprecipitation of nAChRs labeled by the specific nicotinic agonist cytisine with 
antibodies specific for the alpha4 and beta2 subunits, Flores et al (1992) also show that 
high affinity binding sites are composed a402 receptors and moreover that these sites are 
upregulated after chronic exposure to nicotine. The highest densities of mAb 270 labeling 
are in interpeduncular nucleus, the nuclei of the thalamus, superior colliculus, medial 
habenula. More moderate labeling occurs in the presubiculum, layers I and HI/TV of 
cerebral cortex and in the substantia nigra pars compacta/ventral tegmental areas. This 
distribution corresponds quite well with the previously described distribution of high 
affinity nicotinic agonist binding sites (Clarke et al, 1985). It is now apparent that beta2 
has the most widespread distribution of the neuronal nicotinic receptor subunits and can 
coassemble with alpha2, alpha3 or alpha4; however, the majority of this labeling most 






18 

likely represents the expression pattern of receptors containing the alpha4 and beta2 
subunits. It may be the case that a significant proportion of these receptors coassemble 
with the alpha5 subunit in the mature brain (Conroy and Berg, 1998). More recent studies 
examining the brains of beta2 knockout mice show a complete lack of high affinity nicotine 
binding sites providing further evidence consistent with the idea that the most prevalent 
high affinity nAChRs in brain are [32-containing (Picciotto et al., 1995). Interestingly, 
residual binding of other more subtype-specific ligands can be detected in the brains of 
beta2 knockout mice (Zoli et al., 1998) suggesting that there exist high affinity nicotinic 
subtypes in which beta2 does not participate. Electrophysiological studies have also 
indicated that neurons of the habenulo-interpeduncular system have responses consistent 
with expression of a variety of nAChR subtypes (Mulle et al, 1991). 
Low Affinity Brain nAChRs 

The second grouping of brain nAChR is termed low affinity because the desensitized 
state of this receptor does not exhibit a high affinity for agonist. Thus, in binding 
experiments in which the desensitized state of nAChR predominates, this class of receptor 
appears to have a very low affinity for agonist compared to the class of neuronal nAChR 
described above. In contrast, the low affinity brain nAChRs are characterized by a high 
affinity for the binding of a-BTX. The binding of a-BTX is high in cerebral cortex (layers 
I and VI), superior and inferior colliculus, hypothalamus and hippocampus (Clarke et al., 
1985). The only regions of significant overlap with high affinity agonist binding are in 
layer I of cortex and superior colliculus. Until recently, the description of a functional 
nicotinic receptor underlying brain a-BTX binding was problematic because, although 
labeling of protein by a-BTX in brain and peripheral ganglia is readily apparent, it is 
difficult to detect a-BTX sensitive responses in brain and application of a-BTX does not 
inhibit the most easily detected nAChR responses on ganglionic neurons. However, with 
the cloning of the alpha7 subunit it has become apparent that the labeling by a-BTX does in 
fact represent the distribution of functional neuronal nAChRs. 



19 

Affinity purification of high affinity a-BTX binding proteins from chick brain allowed 
N-terminal protein sequencing (Conti-Tronconi et ai, 1985). From the this sequence, 
Schoepfer et al ( 1990) prepared degenerate oligonucleotides which isolated clones 
associated with a-BTX binding. Working independently, Couturier et al. (1990) were able 
to demonstrate that the cDNA encoding a-BTX binding protein from chick brain expresses 
a functional low affinity nicotinic receptor that is sensitive to block by a-BTX and exhibits 
rapid desensitization. This finding was later confirmed by Seguela et al. (1993) for the 
alpha7 clone from rat brain. With the development of rapid agonist application systems, a 
number of investigators have demonstrated the existence of rapidly desensitizing nicotinic 
responses both centrally and peripherally. Thus, it is now clear that the a-BTX labeling in 
rat brain is associated with the alpha7 subtype of nAChR whereas in chick brain the alpha8 
subunit also contributes. The related low-affinity nAChR subunit alpha9 also forms 
homomeric receptors which are blocked by a-BTX but has a very limited distribution 
(expressed in the cochlea for the most part) and recognizes nicotine only as an antagonist 
(Elgoyhenefa/., 1994). 

Functional Roles of Neuronal nAChRs 

Interest in the function of neuronal nAChRs stems from the observation that nicotine can 
increase performance in some measures of memory. Although this effect is well 
documented, it is most likely the case that memory performance is also linked to the state of 
arousal of the test subject. Thus, the effects of nicotine may be mediated through 
nonspecific effects on arousal. However, the additional observation that there is a selective 
loss of cholinergic neurons during the progression of Alzheimer's disease also implicates 
nicotinic systems in the process of memory formation. Nicotinic receptors have also been 
linked to the motor deficits associated with Parkinson's disease, the sensory gating deficits 
associated with schizophrenia and, of course, to nicotine addiction. Furthermore, a 
number of recent reports have described the utility of nicotinic agonists as non-opioid 






20 



analgesics. With these observations in mind, a number of laboratories have proceeded to 
characterize the function of neuronal nAChRs (Holladay etal, 1997). 

The heterogeneity of neuronal nAChR in terms of subunit composition may encode a 
corresponding functional diversity which is required for neuronal function (for review see 
Papke, 1993). It has been demonstrated that the time-course of desensitization and single 
channel kinetics of heterologously expressed alpha3-containing receptors depends on the 
particular beta subunit with which the alpha subunit is expressed (Papke and Heinemann, 
199 1). It is possible that the pattern of activity of individual receptor subtypes could be 
important for coincidence detection in the brain. However, it must also be noted that 
neuronal nAChRs expressed in heterologous expression systems do not fully recapitulate 
the characteristics of native receptors {e.g., in terms of single channel conductance, 
(Sivilotti etal. 1997)). It is unclear whether the differences between heterologously 
expressed receptors and native receptors arise because of the potential for complex subunit 
arrangements in vivo that are not reproduced in heterologous expression systems or 
possibly because of differences in modulation of receptor function by different cell types 
{e.g., by phosphorylation). 

It is also the case that calcium permeability may be dependent upon subunit 
combination. In whole-cell recordings of transfected cells in conjunction with fluorescence 
imaging of a calcium indicator dye, Ragozzino et al (1998) demonstrate that a3(i4 receptors 
have a slightly higher fractional calcium conductance than alpha4-containing receptors. 
However, the bulk of work in this field indicates that any differences in calcium 
permeability across the heteromeric subtypes of neuronal nAChRs are most likely minor. 
By measuring reversal potential shifts with different concentrations of extracellular calcium 
in the cut-open oocyte system (allowing access to both sides of the oocyte membrane), 
Costa et al (1994) demonstrated that heterologously expressed a3(34 receptors have a 
pCa/pNa ratio of 1 . 1 compared to a pCa/pNa ratio of 0. 12 for muscle-type nAChR, while 
other heteromeric subtypes have a permeability ratio in the range of 1.0-1.5. For this class 



21 

of receptors, a direct effect of external calcium on open probability has also been described 
(Amador and Dani, 1995). This direct effect of external calcium may also prove to be of 
physiological significance. By way of contrast, the alpha7 homomeric nAChR has 
pCa/pNa ration in the range of 20 (Seguela et al, 1993). Additional studies of the 
permeability of heterologously expressed alpha7 receptors, in which barium was 
substituted for calcium to minimize any contaminating influence of calcium-activated 
chloride currents to the measurements, indicate a pBa/pNa ratio of 17 (Sands et al, 1993). 
However, it should also be noted that similar measures for a-BTX-sensitive responses in 
hippocampus indicate a pCa/pCs ratio of 6.1 (Castro and Albuquerque, 1995). In the same 
study, the pCa/pCs ratio for the highly calcium-permeable NMDA subtype of glutamate 
receptor was measured to be 10.3. The high calcium permeability of the alpha7 subtype 
may be prove to be of major importance as this receptor subtype could provide a route for 
calcium entry at negative potentials for which both voltage-gated calcium channels and 
NMDA receptors would be inactive. 

The function of nAChR of the peripheral ganglia may be similar to that of muscle 
processing at the level of the ganglion. However, the functional organization of the 
ganglionic synapse has proven to be quite elaborate. The most well characterized 
ganglionic synapse is that of the chick ciliary ganglion. Accessory motor neurons of the 
chick midbrain send processes to this ganglion which terminate in calycal boutons 
enveloping the post-synaptic cell. The post-synaptic receptors at this synapse are of two 
major types: those labeled by mab35 (monoclonal antibody specific for the alpha5 subunit) 
and those bound irreversibly by a-BTX. The mab35 labeled receptors most likely 
correspond to different classes of alpha3-containing receptors (with alpha5) while the a- 
BTX subtype most likely corresponds the alpha7-containing receptors (discussed in more 
detail above). It has been clearly demonstrated that the a-BTX-sensitive subtype can raise 
intracellular calcium and function as rapidly desensitizing nAChRs (Vijayaraghavan et al, 
1992). Studies examining the distribution of these two receptor classes in ganglionic 



'22 

neurons show that alpha7-containing receptors outnumber mab35 labeled receptors and 
seem to be localized in clusters with a perisynaptic distribution. Although not strictly 
necessary for transmission through the ganglion, the responses of alpha7-containing 
receptors seem to contribute a large portion of the synaptic current (Ullian, 1997; Zhang et 
ai, 1996). In consideration of the high calcium permeability of the alpha7 subtype, this 
observation may imply that there exist functional roles in the ganglion for this class of 
receptor apart from participating directly in synaptic transmission. In fact, the regulation of 
intracellular calcium concentration for ganglionic neurons seems to be exceedingly 
complex. Increases in calcium concentration in the post-synaptic ganglionic neuron can be 
initiated by activation of nicotinic or muscarinic acetylcholine receptors (Rathouz etal, 
1995). Activation of muscarinic receptors results in oscillatory increases in intracellular 
calcium that are dependent upon the release of calcium from intracellular stores and coupled 
to phosphatidylinositol turnover while activation of nicotinic receptors results in sustained 
calcium increases dependent upon extracellular calcium. Differences in concentration 
dependence and receptor localization between muscarinic and nicotinic receptors and 
between classes of nicotinic receptor may allow for selective activation of distinct second 
messenger cascades by each of these processes. 

Additional studies of receptor distribution on mature ganglionic neurons by laser 
confocal microscopy indicate that only about 10% of the mab35 labeled receptors lie 
directly across from presynaptic active zones (as labeled by the synaptic vesicle antigen 
SV2) (Wilson Horch and Sargent, 1995). Thus, according to this study the bulk of both 
receptor classes on chick ciliary ganglion neurons are located perisynaptically. It may be 
the case that the nonsynaptic receptors are activated in a long range fashion by the diffusion 
of ACh out of the synaptic cleft. However, the presence of acetylcholine esterase in the 
cleft makes this route of activation by acetylcholine seem inefficient for transmission 
(Zhang etal., 1996). Although the functional significance of perisynaptic receptors (of 
both classes) for transmission dependent upon acetylcholine remains unclear., it is possible 



23 

that activation of perisynaptic alpha7 receptors by choline could influence cellular 
excitability. To complicate matters further, it has been shown that the post-synaptic 
neurons produce arachidonic acid in an activity- and calcium-dependent fashion and that 
arachidonic acid can inhibit alpha7-containing receptors raising the possibility of retrograde 
effects on the presynaptic cell (Vijayaraghavan et ai, 1995). Moreover, the presence of 
functional presynaptic cc-BTX sensitive nAChRs on the presynaptic terminals has recently 
been demonstrated (Coggan et al, 1997). Interestingly, the responses of these pre- 
synaptic a-BTX sensitive nAChRs do not appear to be rapidly desensitizing. This result 
may imply a different subunit composition or different modulation between a-BTX- 
sensitive nAChRs of the pre- and post-synaptic neuron. However, desensitization of 
alpha7 receptors is strongly dependent upon concentration and it may be the case that 
solution exchange at the presynaptic terminal was not complete such that the effective 
concentration reaching the receptors was lower than the bath concentration of agonist in the 
recording chamber. 
Function of Central Neuronal nAChRs 

Brain nAChRs have been implicated in the pathology of a number of disease states 
including Alzheimer's disease, Parkinson's disease and schizophrenia. A number of 
biochemical studies in synaptosomal and slice preparations have shown that nicotinic 
agonists can affect the release of neurotransmitters including 5-HT. dopamine (DA) and 
noradrenaline (NE) implying a potential contribution of presynaptic nAChRs (for review 
see (Wonnacott, 1997)). However, until recently there was scant electrophysiological 
evidence for a functional role of brain nAChR. Consistent with the biochemical data, an 
increasing amount of electrophysiological evidence points to the fact that although somatic 
responses to nicotinic agonists have been recorded, the major functional contribution of 
brain nAChRs arises from a presynaptic receptor population (for review see (Role and 
Berg, 1996)). In general, studies conducted in various synaptosomal and slice preparations 
are consistent with a contribution of heteromeric nAChRs (e.g., cc4p2 or a3p4) to the 



24 

stimulation of neurotransmitter release because the effects are not inhibited by a-BTX or 
the competitive antagonist of alpha7 receptors MLA (methllycaconitine). However, it is 
unclear if an effect of alpha7 receptors could be detected in these studies because of the 
rapidly desensitizing nature of alpha7 responses. 

The medial habenula (MHB) and interpeduncular nucleus (IPN) are connected via the 
fasciculus retroflexus and each of these regions show high expression of a variety of 
nicotinic mRNAs and the presence of nicotine binding sites. In addition, 
electrophysiological characterization of the response of rat MHB neurons indicates the 
presence of at least two distinct subtypes of nAChR (Connolly et al., 1995). Effects of 
presynaptic nAChRs on the responses of post-synaptic IPN neurons have been described 
in separate studies. 

Mulle etal. (1991) characterized the nAChRs of isolated rat MHB and IPN neurons and 
showed a differential rank order potency for agonist activation between the two regions. 
The pharmacological profile of MHB nAChRs is most consistent with the a3[}4 subtype 
while that of IPN neurons is most consistent with alpha2-containing receptors. In addition, 
it was demonstrated that the presence of nicotine reduces the amplitude of afferent volleys 
stimulated in the fasciculus retroflexus. These investigators attribute this reduction to a 
shunting effect as a result of depolarization of the presynaptic terminal upon activation of 
presynaptic nAChRs in the presence of nicotine. Consistent with the pharmacological 
profiles of somatic nAChRs, this effect is insensitive to a-BTX and sensitive to other 
inhibitors of neuronal nAChRs such as hexamethonium and mecamylamine indicating that 
it is most likely mediated by heteromeric nAChRs. 

Further characterization of presynaptic nAChRs via whole-cell recording of neurons in 
slices of rat IPN have demonstrated that nicotine increases the frequency of GABAergic 
and glutamatergic post-synaptic events (Lena et al, 1993). This effect was found to be 
TTX-sensitive and, based on the fact that the frequency increase seemed to be dependent 
upon activation of voltage-gated sodium channels, it was hypothesized that presynaptic 



25 

nAChRs may be located "preterminally". Furthermore, this effect was insensitive to cc- 
BTX but sensitive to traditional inhibitors of neuronal nAChRs such as hexamethonium, 
mecamylamine and DHpE (dihydro-beta-erythroidine). GAB Aergic innervation of IPN 
neurons is thought to arise from local IPN interneurons rather than via the fasciculus 
retroflexus so activation of this population of presynaptic nAChRs may provide a method 
for modulating a local inhibitory circuit. 

In addition to the above results, McGehee et al. (1995) have demonstrated an a-BTX 
sensitive effect of presynaptic nAChRs in cocultures of chick MHB and IPN neurons. In 
these cultures, application of nicotine enhances both evoked and spontaneous release 
(EC50-120 nM) in the presence of TTX indicating that nAChRs are most likely located 
directly on the terminals. The EPSCs were shown to be sensitive to CNQX, a specific 
inhibitor of the non-NMDA subtype of ionotropic glutamate receptors, indicating that 
transmission at this synapse is glutamatergic. The half-maximal inhibitory concentration 
for a-BTX inhibition of the nicotine effect on synaptic transmission was about 70 times 
higher than reported IC50S for the homomeric alpha7 receptor indicating that the nAChRs 
mediating the effects in this study may possibly be heteromeric alpha7 -containing 
receptors. 

Although the effects presynaptic nAChRs in the habenulo-interpeduncular tract have 
been the most well characterized, functional effects via putatively presynaptic nAChRs have 
been described in rat prefrontal cortex, rat hippocampus, rat dorsal raphe nucleus, mouse 
thalamus (ventrobasal complex and dorsolateral geniculate nucleus), chick lateral spiriform 
nucleus (part of the avian basal ganglia) and chick lateral geniculate nucleus. Nicotinic 
receptors in prefrontal cortex seem to modulate excitatory transmission via non-NMDA 
glutamate receptors in a neuronal BTX-sensitive but oc-BTX-insensitive manner (Vidal and 
Changeux, 1993). Presynaptic nAChRs of the dorsal raphe nucleus seem to modulate 
release of both NE and 5HT to metabotropic (G-protein coupled) receptors in an MLA- 
sensitive and MLA-insensitive manner respectively (Li etai, 1998). Release of GABA in 



26 

the thalamus was also found to be modulated by presynaptic nAChRs (Lena and 
Changeux, 1997). In different sensory nuclei, presynaptic effects either required the 
simultaneous activation of voltage-gated calcium channels or could be mediated directly by 
calcium influx through nAChRs. Furthermore, these effects were absent in 02 knockout 
mice implicating presynaptic a4(52 receptors. Presynaptic nAChRs in chick ventrolateral 
geniculate nucleus (LGN) modulate the release of both GAB A and glutamate in an a-BTX 
sensitive and a-BTX insensitive manner respectively; however, the a-BTX sensitive 
effects on glutamate release in chick LGN were also found to be MLA insensitive indicating 
an nAChR of previously undescribed pharmacology (Guo etai, 1998) . Nicotinic 
facilitation of GABA release in chick lateral spiriform nucleus is DHfJE sensitive but 
sensitivity to a-BTX was not tested (McMahon et al, 1994). Consistent with the 
involvement of nicotinic systems in a variety of cognitive disorders, these studies 
demonstrate the potential involvement of multiple subtypes of presynaptic nicotinic 
receptors in the activity of a variety of other neurotransmitter systems ranging from the 
major excitatory and inhibitory ligands for central ionotropic receptors to activators of 
metabotropic neurotransmitter systems. Although functional roles for presynaptic receptors 
are likely specific to each neurotransmitter system in which they are expressed, it is almost 
certainly the case that the presence of presynaptic nAChRs increases the spatial and 
temporal range of inputs which may result in neurotransmitter release and thereby increases 
the receptive field of the post-synaptic neuron. Moreover, the presence of presynaptic 
nAChRs may allow for release independent of a requirement for depolarization to a 
potential which would activate voltage-gated calcium channels. Both of these effects may 
provide a mechanism whereby the probability of release and thus efficiency of a particular 
synapse cam be modulated. 

In view of the importance of hippocampus for memory consolidation and the 
demonstration of multiple forms of synaptic plasticity within hippocampus, elucidation of 
the function of neuronal nAChRs within this circuit is particularly exciting. Whole-cell 



27 

recordings of pyramidal neurons in hippocampal slices indicate that application of nicotine 
increases the frequency of EPSCs at mossy fiber-CA3 synapses (Gray et ai, 1996). 
Consistent with the studies in MHB-IPN cocultures, this effect was found to be a-BTX 
and MLA-sensitive indicating that this effect is mediated via alpha7-containing receptors. 
Moreover, calcium imaging indicates that application of nicotine in the mossy fiber region 
induces similar amounts of calcium influx as invasion of an action potential to the terminal 
region. Therefore, calcium entry directly through nAChRs without the requirement of a 
contribution of voltage-activated calcium channels may be sufficient to facilitate the release 
of glutamate at this synapse. 

In addition to the presynaptic role for nAChRs in hippocampus, it may be the case that 
nAChRs mediate synaptic transmission in the hippocampus directly. There is cholinergic 
innervation of the hippocampus via the septum and high levels of a-BTX binding are 
present in hippocampus. Moreover, robust a-BTX sensitive responses to nicotinic 
agonists are present on cultured hippocampal neurons (Alkondon and Albuquerque, 1993; 
Alkondon et ai, 1994; Castro and Albuquerque, 1995; Zorumski et ai, 1992). In 
addition, several recent studies report the existence of rapidly desensitizing, a-BTX- 
sensitive responses on interneurons but not pyramidal cells of rat hippocampus (Frazier et 
ai, 1998; Jones and Yakel, 1997). 

The demonstration of the existence of functional presynaptic nAChRs raises a question 
as to the proximity of release sites for acetylcholine to presynaptic terminals. In principle, 
activation of presynaptic receptors could arise via at least three mechanisms: direct axo- 
axonic cholinergic synapses, diffusion of synaptically released ACh to nicotinic 
autoreceptors or synaptic spillover of acetylcholine between adjacent synapses. As there 
seem to be few, if any, purely nicotinic responses in brain, it is most likely that the first of 
these options will prove to underlie activation of presynaptic brain nAChRs. The 
demonstration of modulation of evoked neurotransmitter release via stimulation of intact 



28 

nicotinic axo-axonic terminals should provide definitive evidence for the functional 
significance of presynaptic nAChRs. 

However, in the absence of such direct evidence, it is interesting to speculate on 
alternative roles for nicotinic receptors in the brain. The observation that choline is a fully 
efficacious agonist for alpha7 receptors provides a challenge to the notion that the major 
function of this receptor subtype is to participate in fast synaptic transmission (Papke etal, 
1996). As choline would be predicted to be fairly ubiquitous in brain, activation by choline 
may provide an alternate, non-synaptic route by which neuronal calcium influx may be 
regulated. Nonetheless, in light of recent progress in the description of the function of 
nicotinic receptors in brain, it should be noted that both alpha7 knockout mice and beta2 
knockout mice survive to adulthood without any severe anatomical or behavioral 
abnormalities. This result may imply that a high degree of functional redundancy exists in 
cholinergic brain systems such that knockout of a single receptor population is readily 
compensated for (Orr-Urtreger et al., 1997; Picciotto et al, 1995). 

Sensitivity to Noncompetitive Inhibitors 

In addition to differences in distribution and function, neuronal and muscle nAChRs 
differ in their sensitivities to certain classes of noncompetitive inhibitors. These 
compounds are known as ganglionic blockers and show selectivity for the inhibition of 
neuronal nAChRs. The ganglion blocking activity of compounds such as mecamylamine, 
hexamethonium, chlorisondamine, TMP (2,2,6,6 tetramethylpiperidine) and PMP 
(1,2,2,6,6 pentamethylpiperidine or pempidine), has been well documented in the literature 
(Lee etal, 1958; Spinks and Young, 1958). 

The effort to develop pharmaceutical agents specific for neuronal nAChRs dates back to 
the middle of this century. The observation that synaptic transmission through the 
autonomic ganglia was mediated by the chemical messenger acetylcholine led scientists to 
hypothesize that inhibition of ganglionic synapses might be a route by which disorders 



29 

related to autonomic nervous function could be regulated. Since that time, a heterogeneous 
group of compounds with varying selectivities for ganglionic nicotinic receptors has been 
developed and characterized. However, because of the wide range of functions which are 
affected by inhibition of the entire ganglia, clinical applications for these compounds were 
never pursued. In fact, compounds specific for post-ganglionic noradrenergic receptors 
have proven to have a better clinical utility both in terms of safety and effectiveness. 
Although of little clinical utility, these compounds remain of considerable scientific 
importance in part because an understanding of the mechanism of action of pure antagonists 
may allow for a more useful consideration of the observation of mixed agonist/antagonist 
effects of nicotine and other experimental nicotinic agents. 

Although ganglionic blockers have been used extensively as selective blockers of 
neuronal nAChRs, there is a relative paucity of experimental evidence regarding the 
mechanism of selectivity of the various compounds. Each of the compounds seems to act 
in a noncompetitive manner and varying degrees of voltage-dependence for inhibition of 
neuronal nAChRs has been reported. Examination of inhibition of rat submandibular 
ganglion nicotinic receptors by hexamethonium indicate an open channel block mechanism. 
Additionally, recovery from inhibition seems to be dependent upon subsequent application 
of ACh consistent with a model in which hexamethonium becomes trapped in the pore 
(Gurney and Rang, 1984). Consistent with this result, studies of heterologously expressed 
human and rat oc4|32 neuronal nAChRs indicate that inhibition by hexamethonium is use- 
dependent and profoundly voltage-dependent (Bertrand et ai, 1990; Buisson and Bertrand, 
1998). Moreover, analysis of voltage-jump relaxations indicate that inhibition of neuronal 
cx4P2 receptors by hexamethonium is voltage-dependent and relatively long-lived while 
inhibition of muscle nAChRs by hexamethonium occurs independent of voltage (Charnet et 
ai, 1992). 

The ganglionic blocker chlorisondamine (Plummer et ai, 1955) also appears to act via a 
use-dependent mechanism for both cultured ganglionic neuronal nAChRs (Amador and 



30 

Dani, 1995) and striatal synaptosome preparations (El-Bizri and Clarke, 1994). Similar to 
the inhibition of neuronal nAChR by hexamethonium, chlorisondamine inhibition of 
nAChR at the frog neuromuscular junction also seems to exhibit a dependence on 
subsequent application of ACh for recovery consistent with trapping of the inhibitor in the 
ion channel pore (Neely and Lingle, 1986). 

In contrast to the general agreement in the literature about the mechanism of action of 
chlorisondamine and hexamethonium, there is less consensus about the mechanism of 
action of the ganglionic blockers TMP (Spinks and Young, 1958) and mecamylamine 
(Stone et al, 1956). In particular, although mecamylamine is arguably the most widely 
used inhibitor of nicotinic receptors, accounts of the mechanism of action of mecamylamine 
vary greatly. Based on the fact that mecamylamine does not effectively compete with ACh 
or nicotine for nicotinic binding sites, it is surmised that the action of mecamylamine is 
noncompetitive. However, Ascher et al. (1979) conclude that the effects of mecamylamine 
are largely competitive based on the dual observations that inhibition decreases at high 
agonist concentrations and that inhibition appears for the most part voltage-independent. 
Similarly, Bertrand et al (1990) report a relatively long-lived block of a4(32 receptors by 
mecamylamine which appears voltage-independent; however, these authors also report that 
inhibition requires the co-application of agonist indicating a noncompetitive effect. Varanda 
etal. (1985) report a strictly noncompetitive inhibition at neuromuscular junction nAChR. 
In addition, the range of reported IC50S for the effects of mecamylamine vary from about 
40 nM (Ascher et al, 1979; Fieber and Adams, 1991) to 1 uM (Connolly et al, 1992). 
There is also some evidence that differences in sensitivity are associated with the particular 
beta subunit expressed. Specifically, Cachelin and Rust (1995) report an increased 
sensitivity of beta4-containing receptors relative to beta2-containing receptors. Studies 
examining mecamylamine inhibition of heterologously expressed a3|J4 receptors in this 
laboratory indicate that inhibition is strongly voltage-dependent (Webster etal, 
unpublished observations). 



31 

The ganglionic blockers TMP and PMP were originally developed as more potent and 
less toxic alternatives to mecamylamine for the treatment of hypertension (Spinks and 
Young, 1958). However, as more effective adrenergic blockers were developed soon 
after, extensive characterization of this class of blockers did not occur. The observation 
that an analogue of TMP, bis-TMP-10 or BTMPS, used as a light and radiation stabilizer in 
medical plastics, functions as an extremely potent and selective use-dependent inhibitor of 
neuronal nAChRs and a less potent inhibitor of voltage-gated calcium channels has 
renewed interest in the TMP family of ganglionic blockers (Glossmann et al, 1993; Papke 
et al, 1994). Bis-TMP-10 is a member of the bis-TMP-n series of compounds which 
share a common structure consisting of a symmetrical diester of tetramethyl piperidinol 
rings linked by an aliphatic diacid chain containing n carbons (Figure 1-1). Neuronal 
nAChRs exhibit prolonged inhibition after co-application of BTMPS with ACh while 
muscle nAChRs recover completely within five minutes. Both subtypes exhibit only very 
short-term inhibition in response to co-application of the monofunctional inhibitor TMP 
with ACh indicating that the presence of two piperidinol rings may be critical for the 
conversion from short-term inhibition to long-term inhibition. The inhibition by bis-TMP- 
10 exhibits an IC50 of about 200 nM for the open state of heterologously expressed 03(34 
receptors. Substitution of a neuronal beta subunit for the muscle betal subunit increases 
the time course of recovery from inhibition consistent with a role for the beta subunit in 
determining sensitivity to long-term inhibition (Papke et al, 1994). 

Insight into a possible basis for the selectivity of ganglionic blockers may be gained 
from consideration of a previously described mechanism for inhibition of muscle nAChRs. 
Neher and Steinbach (1978) were able to demonstrate that the effects of application of the 
charged local anesthetic derivatives of lidocaine, QX-222 and QX-314, on the responses of 
muscle nAChR to suberyldicholine were consistent with a sequential channel block 
scheme. When these inhibitors are coapplied with ACh, the square pulses of current 



32 





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33 

normally observed for single channel openings are divided into smaller bursts as a result of 
blocking events. The time-course of these bursts are as long or longer than the mean 
channel open time for responses in the presence of agonist alone. This result implies that, 
for these inhibitors, both the binding and unbinding of inhibitor is specific for the open 
state of the channel. Furthermore, inhibition by these compounds is voltage-dependent 
providing further evidence that they act at a site within the ion channel pore. It should be 
noted that there may also be lower affinity sites for the binding of these inhibitors, as the 
utility of the sequential channel block scheme for describing inhibition by QX-222 is not 
maintained at high inhibitor concentrations (>40 uM) (Neher, 1983). 

As inhibition by hexamethonium and chlorisondamine is consistent with open-channel 
block, it follows that sensitivity to inhibition by these compounds may be determined by 
sequence within the pore-lining region of the channel itself. However, the mechanism of 
inhibition of mecamylamine and TMP-related compounds has been described less 
completely. It may be the case these compounds also function as open channel blockers. 
Alternatively, it may be the case the specificity of these ganglionic blockers arises via 
sequence elements located outside the pore-lining domains and these compounds function 
as either allosteric inhibitors or state-dependent, voltage-independent inhibitors. 
Consideration of these possibilities requires a more detailed discussion of the structure of 
nAChR. 

Structure of nAChRs 

Ligand-gated ion channels (LGICs) can be thought of as modular proteins consisting of 
three major components each with an associated high affinity site for the binding of ligands: 
1) the receptor portion which contains the agonist binding sites and is located 
extracellularly, 2) the ion channel portion which contains a high affinity site for the binding 
of noncompetitive inhibitors and spans the cell membrane and 3) the intracellular domain 
which is essential for functional regulation by second messengers and for cytoskeletal 






34 

interactions. Muscle-type nAChRs are the most well characterized of the LGICs. As most 
information about the structure of neuronal nAChR is based on the extensive studies of the 
muscle-type nAChR, structure of muscle nAChR will be considered first and comparisons 
to neuronal nAChR will follow. 

General Structural Features 

Imaging of the nAChR shows the five subunits of nAChR organized symmetrically 
around a central pore (Unwin, 1993). The receptor consists of a vestibule about 30 A in 
width which narrows as it spans the membrane to a diameter of about 8-10 A and 
subsequently widens again at its intracellular extent. The length of the receptor is about 

o 

120 A with the larger portion situated extracellularly while the intracellular portion only 
extends about 15 A into the cytoplasm (Figure 1-2A). The ordering of the subunits around 
the pore remains a matter of some controversy. While it has been shown conclusively that 
the alpha subunits are adjacent to the delta and gamma subunits to form the agonist binding 
sites of muscle-type nAChRs (Blount and Merlie, 1989; Pedersen and Cohen, 1990; Sine, 
1993; Sine and Claudio, 1991), the orientation of the beta subunit with respect to the alpha- 
gamma and alpha-delta pairs is less well characterized. On the basis of electron microscopy 
of biotinylated neurotoxin binding to the alpha subunits and the study of induced P-P and 
native 5-5 dimerization of Torpedo receptors, Karlin (1983, 1995) and Machold (1995) 
proposed that the muscle beta subunit lies adjacent to the delta subunit while the receptor 
models of Unwin ( 1995) place the beta subunit between the two alpha subunits. However, 
the latter proposed subunit arrangement poses a symmetry problem with regards to the 
polarity of the alpha subunit interfaces with gamma and delta subunits. It seems likely that 
the sidedness of the alpha subunit interaction with the gamma or delta subunit would 
remain consistent around the receptor as it would be presumed that very specific 
intersubunit contacts are required for the formation of a functional agonist binding site. 





















Figure 1-2. Cartoons depicting receptor structure (A) and putative membrane topology 
(B) for nAChRs. In each case, the encircled area denotes the putative location of the 
sequence elements which are the focus of this work (TM2: second transmembrane 
domain; ECL: extracellular loop). 

The receptor o dimensions are taken from Unwin, 1993, The nicotinic acetylcholine 
receptor at 9 A resolution. J. Mol. Biol., 229(4): p. 1101-24. The cartoons are the work 
of Dr. Roger L. Papke. 



36 



A 



SYNAPTIC CLEFT 

00000000 



00000000 

CYTOPLASM 




60 a 



0000D 



000000GD. 



- 120 a 



40 a 



20 a 



B 



SYNAPTIC CLEFT 




uu 



TM2 




37 

Each subunit of the pentameric muscle nAChR shares a characteristic configuration (for 
review see (Karlin and Akabas, 1995)). Hydrophobicity analysis suggests a membrane 
topology consisting a large hydrophilic N-terminal putatively extracellular sequence 
followed by four hydrophobic, putative transmembrane domains, with a large cytoplasmic 
domain between transmembrane domains three and four (Figure 1-2B) (Claudio etai, 
1983; Devillers-Thiery et ai, 1982; Noda et ai, 1983). The results of a number of studies 
are consistent with this proposed topology. The second transmembrane domain of each 
subunit seems to contribute to the lining of the ion channel pore (for discussion, see 
below). It has been presumed that each of the four putative transmembrane domains of 
each subunit would have the form of an a-helix. However, somewhat surprisingly, 
imaging of two-dimensional crystalline arrays of Torpedo nAChR at a resolution of 9 A has 
demonstrated that only the second transmembrane of each subunit is helical while the 
surrounding transmembrane domains seem to form beta structures (Unwin, 1995). 
N-Terminal Domain and Agonist Binding Site 

General structural requirements of a ligand binding site can be inferred from the 
structure of the particular ligand. Relevant features of the ACh molecule include a 
positively charged ammonium moiety and the carbonyl oxygen of the acetyl group 
separated by a distance of about 5.9 A. It can be readily appreciated that the ACh binding 
site might include both a negative subsite and a polar subsite with a hydrogen donor 
function separated by an appropriate distance. Most evidence is consistent with this idea 
although the fact that tetramethylammonium (TMA) alone can function as a very low 
efficacy agonist for muscle nAChR and a much higher efficacy agonist for neuronal 
nAChRs (Papke et ai, 1996) indicates that the hydrogen bond acceptor group is not strictly 
required. The agonist binding site of muscle nAChR is comprised of elements contributed 
by each of the alpha subunits with either the gamma or delta subunits such that two distinct 
sites are present on each receptor. Photoaffinity labeling of Torpedo nAChR by the 
competitive antagonist derivative d-tubocurarine (d-TC) yields covalent incorporation into 



38 

each of the alpha, gamma and delta subunits (Pedersen and Cohen, 1990) indicating that 
each of the gamma and delta subunits contributes to a single binding site with a partner 
alpha subunit. However, activation and binding properties of nAChR show a Hill 
coefficient in the range of 2 indicating cooperativity or nonequivalence between 
acetylcholine binding sites. The molecular basis for this nonequivalence is associated with 
the specific subunit contacts of a particular alpha subunit within the pentamer (Blount and 
Merlie, 1989; Sine and Claudio, 1991). Interestingly however, while the a-y interface 
provides the higher affinity site for d-TC, the a-5 interface provides the higher affinity site 
for the agonist carbamylcholine and is presumed to be the higher affinity site for the 
binding of acetylcholine (Sine and Claudio, 1991). 

Three distinct portions of the alpha 1 linear sequence contribute residues that are labeled 
by derivatives of agonists and competitive antagonists (for review see (Changeux etal, 
1992)). It is therefore hypothesized that these regions of alpha subunit linear sequence 
must come together in the mature receptors to contribute to one subsite of an ACh binding 
site. These clusters of contributing residues have been referred to as "loops" by some 
investigators. Specifically, the vicinal cysteine residues Cys-192 and Cys-193 with 
aromatic residues Trp-86, Tyr-93, Trp-149, Tyr-190 and Tyr-198 are believed to 
contribute to one subsite of an ACh binding site. It is interesting to note that all of these 
residues are conserved across muscle and neuronal alphas (with the exception of alpha5) 
and across species (with the exception of human alpha7). It is hypothesized that negatively 
charged and aromatic residues from the delta or gamma subunit {e.g., the homologous 
residues gamma Trp-55 or delta Trp-57) contribute to the second subsite. On a more gross 
level, images of Torpedo nAChR indicate depressions in the alpha subunits surrounded by 
three rods which are presumed to represent a-helices at the approximate level of the ACh 
binding site (Unwin, 1995). 

The activation properties of neuronal nAChR by acetylcholine also exhibit a Hill 
coefficient of about 2 and both alpha and beta subunits contribute to activation by agonists 



39 

and sensitivity to competitive antagonists (Hussy etai, 1994; Luetje and Patrick, 1991; 
Papke et oi, 1993; Papke and Heinemann, 1994). Therefore, it is presumed that, like the 
agonist binding sites of muscle nAChRs, the agonist binding sites of neuronal nAChRs lie 
at the subunit interfaces. Recent studies of the structural requirements for agonist binding 
have taken advantage of the fact that the alpha7 subunit forms a homomeric receptor. 
Chimeric exchanges between the homomeric serotonin receptor (5HT3) and the homomeric 
a7 receptor demonstrate that the necessary requirements for agonist binding are contained 
within the N-terminal 194 amino acids of the alpha7 subunit (Eisele et ai, 1993). 
Additionally, within this stretch of amino acids there are three consensus sites for 
glycosylation. Disruption of these sites by mutagenesis does not affect receptor homo- 
oligomerization or protein surface expression but does affect the expression of functional 
a-BTX binding sites (Chen et al., 1998). Thus, it seems to be the case that receptor 
glycosylation is also an important determinant for the formation of a functional agonist or 
binding site. 

Transmembrane Domains 1-4 (TM1-4) and Cytoplasmic Loop 

From hydrophobicity analysis, it is possible to assess which portions of amino acid 
sequence could potentially contribute to transmembrane domains. However, 
transmembrane domains which function to line the ion channel also contain polar residues, 
the side chains of which are able to interact with permeant ions. Because charged ions in 
solution are partially hydrated, most theories of pore function require the side chains of 
amino acids forming the pore lining to interact with or possibly even substitute for the 
waters of hydration surrounding the permeant ion (Hille, 1992). A full understanding of 
the process of ionic selectivity and permeability will required detailed description of the 
structural elements contributing the pore-lining domain. 

Much of the initial work in describing the three-dimensional arrangement of the putative 
transmembrane domains relied on using photoreactive derivatives of noncompetitive 









40 



inhibitors as photoaffmity labels for the Torpedo receptor and subsequent peptide mapping 
and sequencing. One of the first pieces of evidence that each subunit contributes to a 
common pore-lining domain comes from the observation that, in the presence of agonist, 
the inhibitor chlorpromazine labels residues located on each of the four nonidentical 
subunits of the nAChR and that this labeling can be reduced by application of the 
noncompetitive inhibitor phencyclidine (Oswald and Changeux, 1981). Purification and 
trypsin cleavage of the delta subunit followed by HPLC fractionation of the peptides 
allowed partial peptide sequencing and delta subunit Ser-262 was found to incorporate label 
(Giraudat et ai, 1986). Subsequently, homologous residues in the beta subunit (Ser-254 
and Leu-257 (Giraudat et ai, 1987)), alpha subunit (Ser-248 (Giraudat et ai, 1989)) and 
gamma subunit (Thr-253, Ser-257, and Leu-260 (Revah et ai, 1990)) were also identified 
as incorporating label. All of these residues lie on homologous portions of each subunit in 
a hydrophobic region, the putative second transmembrane domain. Many of the same 
residues are labeled by the noncompetitive antagonist triphenylmethylphosphonium 
(TPMP+) providing further evidence the homologous regions of each subunit contribute to 
a single binding site (Hucho, 1986: Oberthur et ai, 1986). In contrast to these results, 
photolabeling by other NCI derivatives has been incorporated into TM 1 and the 
extracellular loop region between TM2 and TM3. Specifically, an alkylating derivative of 
the desensitizing noncompetitive antagonist meproadifen shows incorporation of label at 
alpha subunit Glu-262, a site predicted to lie in the extracellular loop region between TM2 
and TM3 (Pedersen et ai, 1992) while the photoreactive NCI derivative quinacrine azide 
incorporates within a hydrophobic region corresponding to the putative TM 1 region of the 
Torpedo alpha subunit (Cox et ai, 1985; DiPaola et ai, 1990). 

Additional evidence that the TM2 region of each subunit contributes to the lining of the 
ion channel pore comes from examination of the effects of site-directed mutagenesis on 
channel conductance and the binding of open-channel blockers. By altering the charge of 
particular residues {i.e., mutating negatively charged amino acids to positively charged or 



41 

neutral amino acids) located at homologous positions of the various subunits of Torpedo 
nAChR. Imoto et al. (1988) demonstrated that the rate of ion transport through the channel 
is regulated by three rings of negatively charged and glutamine residues designated the 
intracellular, intermediate and extracellular anionic rings. These rings are situated adjacent 
to the hydrophobic TM2 region as part of the intracellular linker region between TM1 and 
TM2, within TM2 itself and as part of the extracellular linker region between TM2 and 
TM3 respectively. Additionally, mutation of these rings of negative charge can influence 
sensitivity to reduction of current flow by the presence of either extracellular or intracellular 
magnesium (Imoto et al., 1988). The sidedness of the magnesium effect was used to 
confirm the presumed orientation of the receptor with respect to its synaptic and 
cytoplasmic domains. It is also of interest to note that clusters of positively charged amino 
acids adjacent to the internal and external rings of negative charge do not affect channel 
conductance. This observation is consistent with the presumed a-helical structure of the 
TM2 domain because adjacent residues would be predicted to face away from the pore 
region by about 100°. 

Examination of the effects of mutation of residues in the putative TM2 region on the 
binding of open-channel blockers has provided a direct evaluation of the contribution of 
specific amino acids to the lining of the ion channel pore. Based on the voltage-dependence 
of inhibition and analysis of the opening and closing rates of single ion channels of muscle 
nAChR (see discussion above), it was concluded that the quaternary lidocaine derivatives 
QX-222 and QX-314 function as open-channel blockers (Neher and Steinbach, 1978). 
Leonard et al. (1988) were able to verify this conclusion directly by demonstrating that 
mutation of polar residues at homologous sites within the TM2 regions of the muscle 
subunits to nonpolar residues not only decreases channel conductance but also decreases 
the residence time and equilibrium binding affinity of QX-222 for the open state of muscle 
nAChR. This site was designated the inner polar site and is located six amino acids 
downstream (in the linear sequence) from the first residue of TM2 as predicted from 



42 



hydrophobicity analysis (Leonard et al, 1988). Given the proposed nAChR membrane 
topology, this site would lie near the midpoint of the pore closer to the inner mouth of the 
channel. The voltage-dependence of block indicates that the QX-222 binding site 
experiences about 78% of the membrane electric field consistent with a site deep in the 
pore. Subsequent studies demonstrated that similar mutation of polar residues at 
homologous positions on each subunit of nAChR located four residues downstream 
(extracellular) to the inner polar site increases the residence time of QX-222 in the pore 
(Charnet etal, 1990). It was proposed that QX-222 interacts with residues in adjacent 
helices of TM2 via binding of the charged ammonium moiety to residues at the inner polar 
site in conjunction with hydrophobic interactions of the aromatic tail with nonpolar residues 
at a site located more extracellularly in the pore. Based on these studies and the predictions 
from hydrophobicity analysis, a system of nomenclature for the pore-lining region has been 
proposed which numbers the consecutive residues of TM2 from 1' to 20'. According to 
this nomenclature, the inner polar site corresponds to position 6' while the site of 
interaction of the aromatic portion of QX-222 would lie at position 10'. 

More recent studies employing mutation of single residues of interest to cysteine and 
subsequent examination of availability for covalent modification by small, charged 
sulfhydryl-selective reagents, a process known as substituted-cysteine accessibility method 
or SCAM, has provided further insights into accessible residues in both the open and 
closed states of the receptor (Akabas and Karlin, 1995; Akabas et al., 1994; Akabas etal, 
1992; Zhang and Karlin, 1997). These studies have attempted to examine higher-order 
structure of the pore-lining domain. Of particular interest is the location of two elements 
presumed to be critical for ion channel function: the channel gate and selectivity filter. 
From image reconstruction of pseudochrystalline arrays of Torpedo receptor, Unwin 
(1995) hypothesized that a kink in the middle of the TM2 region at the level of a pair of 
highly conserved leucine residues (9' and 10') could function as the channel gate. 
However, SCAM analysis indicates that alpha subunit residues as deep in the pore as E241 









43 

(position -1') are accessible even in the closed state of the channel and that the pattern of 
accessibility of residues throughout TM2 is consistent with an interrupted a-helical 
structure containing a gate at the cytoplasmic end. It may be the case that the interruption of 
the a-helical segment detected by SCAM analysis is analogous to the kink in TM2 detected 
by Unwin. Additional SCAM analysis of alpha subunit TM1 indicates that N-terminal 
residues of this domain may also contribute directly to extracellular mouth of the ion 
channel. 

Electrophysiology studies of receptors incorporating mutant subunits have highlighted 
the importance of highly conserved 9' leucine residues in regulating channel gating (Filatov 
and White, 1995: Kearney et ai, 1996; Labarca etai, 1995). These studies indicate that 
mutation of this residue to a polar residue decreases the EC50 for acetylcholine most likely 
by stabilizing the open state of the channel through polar interactions with the hydrated ions 
of the pore. Similar effects were noted upon mutation of the homologous residue of the 
homomeric a7 receptor (L247T). However, these effects were interpreted in terms of 
reduction in channel desensitization such that the mutant channels exhibit a desensitized but 
conducting state (Revah et «/., 1991). 

Because each family of ion channels exhibits a characteristic ionic selectivity, it is also 
reasonable to hypothesize that particular elements of the pore-lining domain may contribute 
to a selectivity filter which would function to dictate which ions can permeate the channel 
either via steric or electrostatic mechanisms. As discussed above, charged residues have 
been shown to be critical for determining the conductance of the muscle nAChR. 
Somewhat surprisingly however, the presence of charged residues in the pore-forming 
domains alone does not seem to be sufficient to determine the selectivity for anions versus 
cations. By exchanging residues between the cation-selective oc7 receptor and the anion- 
selective GABA A receptor, Galzi et al (1992) demonstrated that insertion of a proline 
residue in the TM1-TM2 linker seems to be critical for converting selectivity from cationic 
to anionic. While receptors incorporating only this insertion are nonfunctional, pairing of 






44 

the proline insertion with mutation of two other pore-lining amino acids to the homologous 
GABA receptor residues produces an anion-selective receptor. Because mutation of these 
residues without insertion of the proline residue in the TM1-TM2 linker was insufficient to 
confer anion selectivity, it may be the case that insertion of the proline alters the orientation 
of the transmembrane helices. It seems likely that the pattern of exposure of amino acids in 
the transmembrane helices together with the character of the exposed amino acids serves to 
regulate selectivity for anions versus cations. 

A closely related question concerns the mechanism by which the permeability to 
different ions of the same charge species is regulated once selectivity for anions versus 
cations has been established. Most studies indicate that a major determinant of ionic 
selectivity of this nature is discrimination on the basis of hydrated ion size. Mutation of 
alpha subunit Thr-244 (position 2') has a large effect on the permeability profile of muscle 
nAChR to large monovalent cations consistent with this region being located near a narrow 
region of the pore and possibly a component of the selectivity filter (Cohen et ai, 1992; 
Imoto etal., 1991; Villarroel etal., 1991). It seems to be the case that permeability to 
divalent cations can be regulated by multiple sequence elements suggesting a more complex 
mechanism for determination of this property than selection on the basis of ion size alone. 
For the highly calcium permeable neuronal a7 receptor, it has been demonstrated that 
mutation of residues located either at the intracellular (E237, position -1') or extracellular 
(L254 or L255, positions 16' and 17' respectively) mouth of the channel can abolish 
calcium permeability. Mutations at position -1' had no other detectable effect on receptor 
function while mutation of residues at positions 16' or 17' seem to effect channel gating as 
evidenced by higher agonist potency and prolongation of response time-course (Bertrand et 
ai, 1993). 

Because the position of the extracellular loop region between TM2 and TM3 in the three- 
dimensional arrangement of the receptor may allow for residues in this domain to play a 
role in transmitting conformational changes from the agonist binding site to the channel 



45 

pore, it has been suggested that this region may contribute to regulation of channel gating. 
In fact, mutation of an asparagine residue in the extracellular loop of homomeric al 
receptors substantially reduces current responses to agonist while binding of a-BTX 
remains intact (Campos-Caro etai, 1996). This residue is conserved across both 
homomer- forming alpha subunits and functional beta subunits of rat and human clones. 
Mutation of the homologous residue in beta4 and expression of the mutant subunit with 
alpha3 yields qualitatively similar results suggesting that this region in structural subunits in 
particular may be important for linking agonist binding to channel activation. 

The role of the intracellular domain between transmembrane domains three and four in 
determining receptor function has also been characterized. This region is the most variable 
across subunits and contains consensus sites for phosphorylation, a finding which has 
suggested a role for differential subunit phosphorylation in determining receptor functional 
characteristics. There is evidence for both serine/threonine phosphorylation and tyrosine 
phosphorylation of muscle n AChRs. The most well described effect of serine/threonine 
phosphorylation is an increase in the rate of desensitization with phosphorylation of the 
gamma and/or delta subunits at a site in the intracellular domain. However, it is unclear if 
this effect is physiologically relevant because of the rapid action of acetylcholine esterase at 
the neuromuscular junction. For neuronal nAChRs. the effects of phosphorylation appear 
to be more heterogeneous. An increase in the response of alpha3-containing receptors of 
chick ciliary ganglion neurons via a cAMP-dependent mechanism has implied that 
phosphorylation of the alpha subunit may convert a population of receptors from a "silent" 
state to a "functionally available" state (Margiotta et al., 1987; Vijayaraghavan et ai, 1990). 
In addition, some recent studies suggest that inhibition of phosphatase activity can increase 
rate of recovery from desensitization (Eilers et al., 1997; Khiroug et al., 1998). These 
results would be consistent with a model in which the desensitized state of the receptor is 
selectively modulated by phosphorylation. However, as these studies involve the 
prolonged application of nicotine rather than the endogenous agonist acetylcholine, it is 



46 

difficult to assess the significance of these findings beyond involvement in pathology 
related to nicotine addiction. 

Relatively few studies have described a specific role for TM3 or TM4 in regulating 
receptor function although it has been widely speculated that these domains mediate the 
interactions of the receptor with surrounding lipid. Chimeric exchanges of TM3 between 
alpha3 and alpha7 subunits and site-directed mutagenesis of residues in TM4 have each 
been shown to have an effect on channel gating (Campos-Caro et ai, 1997; Ortiz-Miranda 
et ai, 1997). However, it is difficult to assign any direct functional implications to these 
effects. 

Relating Structure to Function for Neuronal nAChRs 

It has long been noted that nicotine can increase performance on some measures of 
memory performance. This observation, in conjunction with the fact that a large number of 
cholinergic neurons are lost during the progression of Alzheimer's disease, has led some 
investigators to hypothesize that nicotinic systems may be involved in the processes of 
learning and attention (for review see Levin, 1992). Additionally, brain nAChRs have 
been implicated in the symptomatology of diseases ranging from schizophrenia to nicotine 
addiction. The heterogeneity of potential neuronal nAChR subtypes and their possible 
dysfunction in disease states has provided the impetus for development of subtype-specific 
agonists as candidate therapeutics. However, as is the case for nicotine, many of these 
drugs have both agonist and antagonist effects. In order to define a profile for agonist and 
antagonist specificity, it will be necessary to determine the structural components of the 
neuronal receptor involved in each process. 

This study seeks to expand our knowledge of the structural determinants of sensitivity 
to use-dependent inhibition for neuronal nAChRs. As noted above, bis-TMP-10 is a bi- 
functional analogue of the ganglionic blocker TMP and shows selectivity for the long-term 
inhibition of neuronal nAChRs. The experiments described in this study characterize the 



47 

mechanism of action of bis-TMP-10 and related analogues and, in addition, attempt to 
localize structural determinants of sensitivity to long-term inhibition. It is the ultimate goal 
of these studies to use the knowledge gained from the study of the mechanism of action of 
pure inhibitors to understand the structural basis for the mixed agonism/antagonism 
observed for certain nicotinic experimental therapeutics. 









CHAPTER 2 
METHODS 



Chemicals and Synthesis 

Fresh acetylcholine (Sigma; St. Louis, MO) stock solutions were made daily in Ringer's 
solution and diluted. All other drugs were stored at 4°C at a concentration of 100 mM in 
methanol for a period of no longer than two weeks before use. Bis-TMP-10, bis(2,2,6,6- 
tetramethyl-4-piperidinyl) sebacate (BTMPS), was obtained from Ciba-Geigy (Hawthorne, 
NY), and tetramethylpiperidine was obtained from Aldrich Chemical Company 
(Milwaukee, WI). Bis-TMP-4 (bis (2,2,6,6,-tetramethyl-4-piperidinyl) succinate was 
synthesized by Ciba-Geigy and obtained from Dr. H. Glossmann (Glossmann, et al., 
1993). All other chemicals for electrophysiology were purchased from Sigma Chemical 
Company (St. Louis, MO) or synthesized. Chemicals used for the synthesis were 
purchased from Aldrich Chemical Company. 

Compounds were synthesized by Drs. Kyung II Choi and Benjamin A. Horenstein in 
the laboratory of Dr. Horenstein. To a mixture of 2,2,6,6-tetramethyl-4-piperidinol (3.0 
mmol) and one equivalent of the corresponding ester, unless otherwise specified, in 2 mL 
of dimethyl formamide was added 250 mg of powdered potassium carbonate. The 
resulting mixture was heated at 145-150 °C for 24-72 hrs under a gentle stream of N2- 
After cooling, the reaction mixture was partitioned between water and methylene chloride. 
The organic layer was separated, washed with water and brine, dried (anhydrous MgS04) 
and evaporated to dryness to give a crude product, which was purified via salt formation 
(HC1 or acetic acid), extraction, or column chromatography. Compounds were 
characterized by mass spectrophotometry and high resolution nuclear magnetic resonance. 
All compounds were recrystallized before use in electrophysiology experiments. 



48 



49 

Production of Chimeras and Sequencing 

All chimeric genes were constructed by the method of overlap extension PCR (Horton et 
al, 1989). The beta subunit chimeras were designed and produced in large part by Wayne 
Gottlieb and Ricardo Quintana. The genes encoding the betal, beta4, gamma and delta 
subunits were cloned into p-Bluescript SK-. Specific PCR primers were designed to 
generate mutants exchanging just the bases necessary to code the TM2 or ECL region. 
Each primer contained 27 bases of the sequence flanking the TM2 or ECL sequence on one 
side and 24 bases that coded for the TM2 or ECL region to be exchanged. 
Oligonucleotides were designed to contain a unique silent restriction site in the mutant 
region for future screening, and synthesized by the University of Florida DNA Synthesis 
Core. Separate PCR reactions consisting of the appropriate PCR primer with template and 
either T3 or T7 primer selectively amplified the upstream and downstream portions of the 
gene of interest with overhanging chimeric sequence. These two products were then put 
together in a second PCR reaction with T3 and T7 primers. The region of chimeric 
sequence overlap formed double stranded DNA that primed elongation in both directions, 
and the full length product was amplified using T3 and T7 primers. The region coding for 
mutant sequence was then cut out with restriction enzymes and cloned back into the original 
plasmid, reducing the amount of PCR-generated sequence in the final constructs. Clones 
were evaluated by both restriction analysis and sequencing through the PCR generated 
region either by the dideoxy chain termination method (Sanger et al., 1977) using the 
Sequenase 2.0 kit from United States Biochemical Corporation (Cleveland, Ohio) in the 
laboratory of Dr. Jeffrey K. Harrison or by automated fluorescence sequencing in the 
University of Florida DNA Sequencing Core. 

Preparation of RNAs and Oocyte Expression 

In vitro cRNA transcripts were prepared using the appropriate mMessage mMachine kit 
from Ambion Inc. (Austin, TX) after linearization and purification of plasmids containing 



50 

cloned cDNAs. RNA transcripts were stored at -80 °C as water stocks at a concentration of 
either 200 ng/jil or 600 ng/|il. Concentrations were determined from measures of percent 
incorporation of 32 P-labeled UTP via scintillation counting. 

Ovarian lobes were surgically removed and then cut open to expose the oocytes. The 
ovarian tissue was then treated with collagenase from Worthington Biochemical 
Corporation (Freehold, NJ) for about 2 hours at room temperature (in calcium-free Barth's 
solution: 88 mM NaCl, 10 mM HEPES pH 7.6, 0.33 mM MgS0 4 , 0. 1 mg/mi gentamicin 
sulfate). Subsequently, stage 5 oocytes were isolated and injected with 50 nl each of a 
mixture of the appropriate subunit cRNAs following harvest. Barth's solution was 
changed daily under semi-sterile conditions. Recordings were made 2 to 7 days after 
injection depending on the cRNAs being tested. 

Electrophysiology 
Two-Electrode Voltage Clamp 

Initial recordings were made on a Warner Instruments (Hamden, CT) OC-725C oocyte 
amplifier and RC-8 recording chamber interfaced to a Macintosh personal computer, while 
the majority of experiments employed a Gene Clamp 500 amplifier (Axon Instruments; 
Foster City, CA) interfaced to a Gateway 2000 (N. Sioux City, SD) P5-75 personal 
computer. Comparable results were obtained on both sets of equipment. Initial 
experiments were performed in a configuration such that a 2 ml bolus of drug was applied 
after loading of a loop at the terminus of the drug delivery system, while subsequent 
experiments were conducted in a configuration where drug application was electronically 
controlled and regulated by duration rather than volume, permitting more rapid solution 
exchange without stoppage of flow through the chamber. Oocytes were placed in a Lexan 
recording chamber with a total volume of about 0.6 ml and perfused at room temperature 
by frog Ringers (1 15 mM NaCl, 2.5 mM KC1, 10 mM HEPES pH 7.3, 1.8 mM CaCl 2 ) 
containing 1 uM atropine to block potential muscarinic responses. A Mariotte flask filled 



51 

with Ringers was used to maintain a constant hydrostatic pressure for drug deliveries and 
washes. Drugs were diluted in perfusion solution and applied from a reservoir for 10 
seconds using a 2-way electronic valve. Data were acquired using Axoscope 1 . 1 software 
(Axon Instruments; Foster City, CA) at a 20 Hz sample rate and filtered at a rate of 10 Hz 
using either a CyberAmp 320 external filter (Axon Instruments; Foster City, CA) or the 
filter in the amplifier. The rate of drug application and perfusion was 6 ml/min in all cases. 
Current electrodes were filled with a solution containing 250 mM CsCl, 250 mM CsF and 
100 mM EGTA and had resistances of 0.5-2 MQ. Voltage electrodes were filled with 3 M 
KC1 and had resistances of 1 -3 MQ.. Oocytes with resting membrane potentials more 
positive than -30 mV were not used. 

Cut-Open Oocyte Vaseline-Gap Voltage Clamp 

Experiments were conducted using the modified chamber described by Costa et al. 
(1994) and available commercially from Dagan. Measurements were made using a Dagan 
amplifier interfaced to a Gateway 2000 (N. Sioux City, SD) P5-75 personal computer 
running pClamp 7 software. For most experiments, data were acquired at a rate of 200 Hz 
and filtered at a rate of 20 Hz using the filter in the amplifier. Ringers solution was perused 
to the external face of the oocyte by gravity flow from a Mariotte flask while an internal 
solution consisting of 100 mM KC1, 10 mM HEPES, and 10 mM EGTA (pH 7.4) was 
perfused to the internal face of the oocyte by syringe pump (World Precision Instruments) 
at a rate of O.Olml/hr. Agonist was applied via a modified U-tube controlled by an 
electronic 2-way valve (General Valve Corporation) activated digitally by the computer. An 
insert in the recording chamber produced a region of laminar flow across the exposed 
portion of oocyte membrane in the chamber allowing for relatively rapid application of 
agonist. Internal perfusion pipettes were pulled to a length of about 4.7 cm and broken to a 
diameter of > 100 urn to allow adequate perfusion and reduce series resistance. Internal 



52 

perfusion pipettes were then coated with a mixture a parafilm and mineral oil to prevent 
leaking of solution between the pipette and the floor of the chamber. 

Experimental Protocols and Data Analysis 

For the majority of experiments, current responses to drug application were studied 
under two-electrode voltage clamp at a holding potential of -50 mV unless otherwise noted. 
Holding currents immediately prior to agonist application were subtracted from 
measurements of the peak response to agonist. All drug applications were separated by a 
wash period for a length of time as noted (five minutes in most cases). At the start of 
recording, all oocytes received an initial control application of ACh to which subsequent 
drug applications were normalized in order to control for the level of channel expression in 
each oocyte. An experimental application of ACh with inhibitor was followed by an 
application of ACh alone ten minutes after the control ACh application used for 
normalization. In some receptor subtypes (e.g., a3p4), rundown was observed to stabilize 
after a second application of ACh. For these subtypes, responses were normalized to the 
second of two initial control ACh applications. Means and standard errors (SEM) were 
calculated from the normalized responses of at least 4 oocytes for each experimental 
concentration. 

For all experiments involving use-dependent inhibitors, a concentration of ACh was 
selected sufficient to stimulate the receptors to a level representing a reasonably high value 
of Popen at the peak of the response, while minimizing rundown with successive ACh 
applications. For potent use-dependent inhibitors, this concentration is adequate to achieve 
maximal inhibition (Papke et al., 1994). Specific concentrations for each receptor subtype 
are as noted. 






53 



For concentration-response relations, data were plotted using Kaleidagraph 3.0.2 
(Abelbeck Software, Reading, PA) and curves were generated using the following 
modified Hill equation (Luetje and Patrick, 1991) 



Response = maxl & — H 

[agonist]" + (ec50)" 



where I ma x denotes the maximal response for a particular agonist/subunit combination, and 
n represents the Hill coefficient. I max , n, and the EC50 were all unconstrained for the 
fitting procedures, and the r values of the fits were all > 0.96 (the average r value = 0.97). 

For use-dependent inhibitors, measurements of peak response at the time of co- 
application of agonist with inhibitor underestimate steady-state inhibition in our system. 
Therefore in experiments assessing rate of recovery from use-dependent inhibition, 
inhibitor alone was pre-applied for a length of time sufficient to achieve maximal 
concentration in the chamber prior to application of agonist. This pre-application protocol 
maximized the probability of block upon channel activation as a function of inhibitor 
concentration and permitted the use of peak current as a more accurate measure of steady- 
state inhibition for applications of ACh in the presence of the TMP compounds. After 
normalization to the control response (as described above), total inhibition can be calculated 
by subtracting the normalized value from 1. In this manner, nearly complete inhibition at 
the time of co-application of agonist with inhibitor is observed and a recovery rate from this 
point in time can be estimated. Agonist concentrations were selected in order to minimize 
rundown with successive ACh applications while still providing a high enough probability 
of channel activation during the time-course of a response to achieve close to 100% 
inhibition, and are noted in the figure legend. 



54 
Recovery rate data were fitted by the equation 

% Inhibition = Io(e -t/x ) 

which describes a first order process where I represents the inhibition at time t=0 and x is 
the time constant for recovery. The r values of the displayed fits were all > 0.96 (the 
average r value = 0.98). 

For experiments assessing voltage-dependence of inhibition, oocytes were initially 
voltage clamped at a holding potential of -50 mV and a control application of ACh alone 
was delivered. The holding potential was stepped to +20 mV for 30-60 s prior to co- 
application of either ACh with bis-TMP-10 or ACh alone. Thirty to sixty seconds after the 
peak of the co-application response, voltage was stepped back down to -50 mV and 
residual inhibition was evaluated with two subsequent applications of ACh alone separated 
by 5 minutes. 

For experiments assessing protection from long-term inhibition by application of a short 
term inhibitor, a saturating concentration of the short term inhibitor [either QX-3 14 
(lidocaine N-ethyl bromide) or TMP (2, 2, 6, 6-tetramethylpiperidine)] was applied for 30- 
60 s prior to the application of ACh and the long-term inhibitor (bis-TMP-10). The 
application of the short term inhibitor continued throughout the time -course of the co- 
application of ACh with long-term inhibitor until at least 30 s after the co-application. The 
concentration of inhibitor and period of application was selected to maximize the potential 
for protection effects. Recovery from inhibition was evaluated in 3 minute intervals after 
the application of inhibitor in the case of aipi(P4TM2)y8 receptors and 10 minutes after 
application of inhibitor in the case of a3p4 receptors. 

In experiments using the cut-open oocyte system, data from each oocyte was normalized 
to an initial 5 s application of ACh in the presence of normal internal solution ( 100 mM 
KC1, 10 mM EGTA, 10 mM HEPES, pH 7.4). For experiments evaluating the effects of 



55 

intracellularly applied inhibitors, control of perfusion was then switched to a second 
syringe pump containing inhibitor diluted into the internal solution to a concentration of 2 
uM. Internal solution is pumped (0.01 ml/hr) into the perfusion pipette via the base of the 
pipette holder with an approximate pipette solution exchange time of 10 minutes based on 
the inclusion of a dye in the solution. Experimental measurements for activation in the 
presence of intracellularly applied inhibitor were made in 3 minute intervals starting 15 
minutes after switching to the second syringe pump. 



CHAPTER 3 
RESULTS 



Structural Determinants of Sensitivity to the TMP Family 
of Noncompetitive Inhibitors 



Muscle Delta Subunit Effects 

It has been previously published that a bis- analogue of the ganglionic blocker TMP 
produces long-term inhibition of heterologously expressed a3[}4 receptors while TMP itself 
only produces short-term inhibition (Papke, 1994). One of the main goals of the present 
work is to characterize the basis for this selectivity. Although muscle nAChRs show rapid 
recovery from inhibition by bis-TMP-10 (within five minutes), this compound does 
produce appreciable inhibition of muscle nAChRs at the time of co-application with ACh 
while a3(34 receptors show pronounced inhibition at both time points (Papke et al., 1994); 
Figure 3-1). This observation demonstrates the presence of a high affinity bis-TMP-10 
binding site(s) on neuronal nAChRs and furthermore suggests the presence of relatively 
low affinity site(s) for binding of bis-TMP-10 on muscle nAChR. Moreover, because bis- 
TMP-10 is a bi-functional molecule composed of two TMP moieties linked by an aliphatic 
chain and TMP itself is an effective ganglionic blocker, it may be the case that the time 
course of recovery from inhibition by bis-TMP-10 is determined by the number of available 
TMP binding sites per pentameric receptor. In this case, the long-term inhibition of 
neuronal receptors may be a result of the presence of multiple neuronal beta subunits in the 
receptor pentamer while the short-term inhibition of muscle receptors may be associated 
with the presence of only a single TMP binding site. It has been shown previously that 
expression of either the neuronal beta2 or beta4 subunit with the other muscle subunits 
prolongs the time course of recovery from inhibition by bis-TMP-10. This result may 






56 



Figure 3-1. Long-term inhibition by bis-TMP-10 is specific for neuronal nAChR 
subunit combinations. Representative traces are shown in A while mean data are 
shown in B. A) In each set of traces, the responses on the far left and far right are to 
control applications of 30 uM ACh alone while the middle trace is the response to 
coapplication of ACh with 2 uM bis-TMP-10. All responses are separated by five 
minutes. B) All mean data are expressed relative to the initial control response. Each 
column represents the mean of at least 4 oocytes. Similar results have been published 
previously by Papke etai, 1994. 



58 



A 




600 nA 




300 nA 



alplyS 

I 



a3(34 

~1~ 




B 



1.25 

"o 

E 

c 1 

o l 

o 
£0.75 



-2 



0.5 



u 

C 

EL0.25 



□ a3(34 
0alply5 




30 |iM ACh 
with2(iMbis-TMP-10 



30 \iM ACh alone 
five minutes later 
























59 

imply that a single site for TMP binding is present on the wild-type muscle receptor and 
substitution of a neuronal beta subunit for the muscle beta subunit provides a second 
potential TMP binding site. To characterize structural elements of muscle subunits which 
may contribute to a site for bis-TMP-10 binding, muscle-type receptors lacking either the 
gamma or delta subunit were expressed and characterized according to their sensitivity to 
inhibition by bis-TMP-10. alpl5 and aipiy receptors have been previously characterized 
in a number of studies (Charnet et ai, 1992; Jackson et ai, 1990; Kullberg et ai, 1990; Lo 
etai, 1990) and show concentration-response relationships typical of nAChRs in our 
system as well with EC50S for activation of 35 and 25 uM respectively (not shown). 
Because the agonist binding sites for muscle nAChRs are located at the interface of the 
alpha subunits with the gamma and delta subunits, it is presumed that the receptors formed 
by omission of either gamma or delta subunit RNA contain two copies of the included 
subunit. For example, aipi8 (gamma-less) receptors would have a subunit stoichiometry 
of 2: 1:2. 
Contributions of Delta Subunit to Inhibition by Bis-TMP-10 

Muscle-type and aipiy receptors show nearly complete recovery from inhibition by co- 
application of bis-TMP-10 with ACh within five minutes, while aipi5 receptors display 
prolonged inhibition as measured five minutes after co-application of ACh with bis-TMP- 
10 (Figure 3-2A). The mean response of alpl6 receptors to application of ACh alone five 
minutes after co-application with bis-TMP-10 is 24±03% of the response to the initial 
control application of 10 uM ACh (n=8) while the corresponding responses of aipiy (n=4) 
and wild-type muscle receptors (n=6) are near control levels. Omission of gamma subunit 
RNA seems to have effects on the time-course of recovery from inhibition by bis-TMP-10 
comparable to the effects seen previously after substitution of neuronal beta subunit RNA 
(beta4 or beta2) for muscle beta subunit RNA (betal) (Papke etai, 1994). 



Figure 3-2. The blockade of a(35 nAChRs by bis-TMP-10 (A) and TMP (B). The cluster 
of bars on the left represents the mean peak responses (±SEM) of oocytes to the co- 
application of 10 uM ACh and the inhibitor, while the cluster of bars on the right 
represents the normalized responses to 10 U.M ACh alone after a 5 minute wash period. 
The response of each oocyte was normalized to an initial response to 10 u\M ACh applied 5 
minutes prior to the co-application of ACh with bis-TMP-10. 









61 



A 



1.25 -i 



c 
o 
o 
o 

— 

■J 
> 

— ' 



V3 



1/3 



2|LiM Bis-TMP-10 




1 uM ACh 1 nM ACh alone 

with 2 |iM bis-TMP-10 five minutes later 



B 



^j a(3y5 (Wild-type muscle) 
§3 a(3y (Delta-less muscle) 

K3 tt(38 (Gamma-less muscle) 



4jaM TMP 




1 uM ACh with 10 uM ACh alone 
4 \\M TMP f" lve minutes later 



62 

Contributions of Delta Subunit to Inhibition by TMP 

aipi5 receptors also show increased sensitivity to the monofunctional inhibitor TMP (2, 
2, 6, 6-tetramethylpiperidine) at the time of co-application as compared to wild-type muscle 
and aipiy receptors (Figure 3-2B). The mean response of alplS receptors to co- 
application of 10 \iM ACh with 4 uM TMP is about 60% of the control response to an 
initial application of ACh alone (n=4) while the responses of normal muscle-type receptors 
and aipiy receptors are near control levels. As these data are for a single concentration of 
TMP only, the possibility that the difference in sensitivity observed in these experiments 
simply reflects a shift in the concentration dependence of the effect cannot be ruled out. In 
fact, although an effect of TMP on wild-type muscle receptor is not observed at the 
concentration used in this study, muscle-type nAChRs do exhibit a weak sensitivity to 
short-term inhibition by this compound. The IC50 for short-term inhibition of muscle 
receptors by TMP appears to be in the range of 40 uM, a concentration about 200 times 
higher than the reported IC50 for long-term inhibition of oc3p4 receptors by bis-TMP-10 
(Kabakov and Papke, unpublished observations). Although aipi6 receptors do exhibit an 
increased sensitivity to short-term inhibition by TMP compared to wild-type muscle 
receptors, it is interesting to note that a corresponding sensitivity to long-term inhibition by 
TMP is not observed. Thus, the effects of TMP are strictly short-term in nature and seem 
to be equivalent to the effects of bis-TMP-10 on wild-type muscle or aPy receptors. This 
observation is consistent with the results of a previous study in which neuronal beta 
subunits were substituted for muscle beta subunits in that, for both cases, although 
sensitivity to short-term inhibition by TMP increases with incorporation of a TMP sensitive 
subunit, long-term inhibition only occurs with application of bi-functional TMP 
compounds such as bis-TMP-10. The requirement for a bi-functional molecule may imply 
that a secondary process associated with interactions between the receptor and the aliphatic 
linker or second TMP moiety of bis-TMP-10 contributes to the time course of recovery 
from inhibition. 



63 

Contributions of Delta Subunit to Inhibition by Mecamylamine 

In order to have a context for consideration of the effects of TMP-related compounds, 
similar experiments assessing sensitivity to the ganglionic blocker mecamylamine were 
conducted. Omission of gamma subunit RNA also leads to increased sensitivity to 
inhibition by mecamylamine (Figure 3-3A). Additional experiments assessing the 
contribution of beta subunits to inhibition by mecamylamine indicate that, as expected for a 
ganglionic blocker. a3[}4 neuronal receptors show more pronounced inhibition by 
mecamylamine than any of the other subunit combinations tested (Figure 3-3B). 
Substitution of the neuronal beta subunit for the muscle betal subunit seems to increase 
sensitivity to inhibition by mecamylamine at the time of co-application slightly. However, 
inhibition five minutes after application of mecamylamine was detected only in the case of 
cc3p4 (about 31%) and aipi8 (about 38%) receptors (Figure 3-3B). 
Response Kinetics Imply Mechanism of Inhibition 

Responses of both muscle and neuronal nAChRs to the co-application of bis-TMP-10 
with ACh exhibit a decreased to time to peak response compared to the peak of the control 
response to ACh alone consistent with a dependence on prior activation of the channel for 
inhibition (Figure 3-4). In contrast, responses to the co-application of mecamylamine with 
ACh exhibit a clear decrease in time to peak only for a3(34 receptors. Based on these 
observations, inhibition by bis-TMP-10 appears to be purely-use dependent and relatively 
long-lived for all receptor subtypes whereas, for some subtypes of nAChR, inhibition by 
mecamylamine may occur via multiple mechanisms. The differences in time to peak 
response for the two inhibitors indicate that mecamylamine may not act by a purely use- 
dependent mechanism on non-neuronal receptors, particularly the aipi6 receptor subtype. 
Alternatively, it may be the case that the off-rate of mecamylamine is rapid compared to the 
time-course of drug application so that the decay phase of the macroscopic response is not 
appreciably affected by the presence of the inhibitor. For open-channel blockers with a 
very fast off-rate, it would not be expected that time to peak would be affected appreciably 



Figure 3-3. The blockade of a(3y, a(55, and muscle-type (alplyS) receptors (A) and muscle- 
type, alp4v8 and neuronal (a3f}4) receptors (B) by mecamylamine. The cluster of bars on 
the left represents the mean peak responses (±SEM) of" at least 4 oocytes to the co- 
application of 10 pM ACh and the inhibitor, while the cluster of bars on the right 
represents the peak responses to 10 pM ACh alone after a 5 minute wash period. The 
response of each oocyte was normalized to its response to an initial application of 10 pM 
ACh 5 minutes prior to the co-application of ACh with inhibitor represented in the figure. 















65 




□ aipiyS 
H alply 

□ aipiS 



1 (iM ACh 1 |iM ACh alone 
with 10 |iM mec. five minutes later 




□ alplyS 
B alp4y8 
H a3p4 



[ uM ACh with i o uM ACh alone 
10 uM mec. five minutes later 



Figure 3-4. Inhibitor effects on the kinetics of macroscopic currents. 
Representative waveforms of responses from ocp6 (A and B), wild type muscle (C 
and D), alp4y5 (E and F) and neuronal (G and H) receptors to a pulse of 30 uM 
ACh alone (line 1, gray), and a co-application with either 2 uM bis-TMP-10 
(traces on left) or 10 uM mecamylamine (traces on right) and 30 uM ACh (line 2, 
black). For all the traces, the thin black line (3) plots the inhibited current scaled 
to the same peak value as the control in order to visualize the kinetics of inhibition. 
The thick bars under the traces represent the period of drug application. Note that 
due to the prolonged response of a(38 receptors, the time scale in A and B is 
expanded. 









67 



A Bis-TMP-10: aipiS injected oocyte, J3 Mecamylamine: aipi8 injected 
gamma-less receptor oocyte, gamma-less receptor 

1.— 2 





30 



60 90 

Seconds 



120 



30 



60 90 

Seconds 



120 



Bis-TMP-10: alply5 injected 
oocyte, Muscle-type receptor 



\j Mecamylamine: alply5 injected 
oocyte, Muscle-type receptor 





15 30 45 

Seconds 



60 



E 



15 30 45 

Seconds 



60 



F 



Bis-TMP-10: a Ip4y5 injected oocyte, 17 Mecamylamine: alp4y5 injected 
neuronal/muscle hybrid receptor oocyte, neuronal/muscle hybrid receptor 

2 

\ 





G 



15 30 45 60 

Seconds 

Bis-TMP-10: a3p4 injected JJ 

oocyte, neuronal receptor 



15 30 45 

Seconds 

Mecamylamine: a3P4 injected 
oocyte, neuronal receptor 

2 



60 





15 30 45 

Seconds 



60 



15 30 45 

Seconds 



60 



68 

whereas the falling phase of the response may in fact be prolonged by the presence of the 
inhibitor. However, the observation of long-term inhibition after application of bis-TMP- 
10 with ACh to apS receptors makes it seem unlikely that a rapid off-rate of the inhibitor 
underlies the change in response waveform . In some cases, where inhibition is clearly 
use-dependent, a secondary peak is observed in the waveform of the co-application 
response (Figure 3-4; A and H). Since this secondary peak corresponds exactly with the 
removal of agonist and occurs only in conditions where inhibitor is present and acting in a 
clearly use-dependent manner, it is likely that this peak represents relief from a short-time 
course, low affinity inhibition. Because this inhibition relaxes within the time course of the 
agonist response, it most likely represents an entirely different form of inhibition (or at least 
inhibition with different kinetics) from the relatively long-term inhibition discussed above. 

It is interesting to note that even though muscle receptors show rapid recovery from 
inhibition by bis-TMP-10, the waveform of the response to co-application of bis-TMP-10 
with ACh looks very similar across all receptor subtypes tested. This similarity may 
indicate that the mechanism underlying the initial phase of inhibition by bis-TMP-10 is 
similar across all receptor subtypes while the rate of recovery from inhibition may be 
related to some secondary process associated with an interaction between the inhibitor and 
structural elements specific to individual subunits. Furthermore, the affinity of this 
secondary interaction may be dependent upon the number of TMP-sensitive subunits 
present within a particular receptor subtype. Because inhibition by bis-TMP-10 shows the 
most pronounced selectivity for neuronal nAChRs and also has the longest time-course, 
subsequent investigations focused on inhibition by this compound. 
Time-course of Recovery of a(35 Receptors after Inhibition by Bis-TMP-10 

In order to examine the rate of recovery from inhibition by bis-TMP-10 for aipi8 
receptors, ACh was applied at time points beyond five minutes after co-application of ACh 
with bis-TMP-10 and residual inhibition was evaluated (Figure 3-5). Because in this 
system, peak currents at the time of co-application of bis-TMP-10 with ACh underestimate 



Figure 3-5. Sequence elements on the delta subunit determine the time course of 
recovery from inhbition by bis-TMP-10. A) Representative responses of a(35, aPy and 
aPy(5'-6'8) receptors. For each receptor subtype, the trace at the far left and series of 3 
traces at the far right are responses to ACh alone while the middle trace is the response 
to the coapplication of 30 uM ACh with 2 uM bis-TMP-10. All responses are 
separated by 5 minutes. B) Recovery from inhibition by bis-TMP-10 as a function of 
time. The data points at t=0 minutes represent a measure of total inhibition observed 
after coapplication of ACh with 2 uM bis-TMP-10 (see Methods). Data points at t=5, 
t=10 or t=15 minutes represent mean values of residual inhibition measured after 
application of ACh alone. Since for cclply8 and apy receptors, nearly full recovery was 
effectively achieved after only 5 minutes of wash, the data at the 10 and 15 minute 
time points for these receptor subtypes are omitted for clarity. All drug applications 
are separated by five minute wash periods. The concentration of ACh in each 
experiment is either 10 uM (alplyS) or 30 uM (aipiy, aipi8 and aipiy(5'-6'S)). All data 
points represent the mean responses of at least 4 oocytes and are fit with an equation 
describing exponential recovery (see Methods). 



70 



A 



a(35 



ocPy 







20 nA 



60s 




3|iA 



apy 



(5'-6'8) 




V 



\r T~ 



150 nA 



B 



1 r 



c 
o 
•a 

X) 

1 

i 0.1 

1 
0* 



0.01 




5 10 15 

Time (mins.) 



20 



71 

total inhibition at this time point, even with application of a saturating concentration of bis- 
TMP-10, for these experiments. 2 uM bis-TMP-10 alone was applied continuously for 15- 
20 seconds prior to application of ACh in the continued presence of 2 uM bis-TMP-10. 
Using this protocol, nearly total inhibition at the time of co-application of 2 ^M bis-TMP- 
10 with ACh was observed and the time-course of recovery from this time point could be 
measured by fitting the data with a single exponential. Consistent with the observations 
five minutes after application of inhibitor, al|3l5 receptors exhibit prolonged inhibition with 
a time constant of recovery of about 25 minutes while aipiy receptors recover rapidly from 
inhibition with a time constant of about 2.5 minutes. 

Residues in the Delta Subunit TM2 Region Regulate Time-Course of 
Recovery from Inhibition by Bis-TMP-10 

Based on previous descriptions of residues in the pore-forming domains which 

contribute to the binding of other use-dependent inhibitors, it was hypothesized that 

sequence within the TM2 domain of delta subunits may regulate sensitivity to long-term 

inhibition by bis-TMP-10. In particular, a pair of adjacent residues at positions 5' and 6' 

were found to differ between the gamma and delta subunits (underlined sequence below). 



intracellular MEMBRANE SPANNING II extracellular 

GAMMA CTVTmgVLlAQTvTIjFLVAKK 

DELTA TSVAISVLLAQSVFLLLISKR 

/' 20' 



In the gamma subunit sequence, a threonine residue is at position 5' while an asparagine 
residue occupies position 6'. For the delta subunit, the residues at these positions are 
isoleucine and serine respectively. As position 6' has previously been found to be 
important for regulating the binding kinetics of local anesthetics such as QX-222 and QX- 
314 (Leonard et ai, 1988), it is likely that amino acids at this position face the ion channel 
pore. Therefore, it seems reasonable to speculate that, if bis-TMP-10 acts as an open- 
channel blocker, residues at this position may regulate the time-course of inhibition of by 






72 

this drug also. Additionally, it may be the case that the size of the adjacent residue also 
influences exposure of the amino acid. Towards testing these hypotheses, a pair of mutant 
cDNAs were constructed which exchange the bases coding for these two amino acids 
between the gamma and delta subunits. In order to assess the effects of these exchanges on 
the time-course of inhibition by bis-TMP-10 without the influence of a wild-type gamma or 
delta, these mutant subunits were then expressed individually with wild-type muscle alpha 
and beta subunits only. Receptors including the delta^^' gamma) double mutant either do 
not express sufficiently or are nonfunctional in this configuration. However, receptors 
including the gamma mutant subunit (oclplY(5'-6'5) receptors) give robust responses to 
application of ACh within 5-6 days after injection and exhibit concentration-response 
relationships typical of nAChRs. However, this class of mutant receptors a higher EC50 
for activation compared to aPy or ap5 receptors in the range of 135 uM (not shown). Also, 
aipiy(5'.6'8) receptors show about 70% inhibition five minutes after application of 30 uM 
ACh with 2 uM bis-TMP-10 (Figure 3-5 A) and recover from inhibition with a time 
constant of recovery of about 10 minutes (Figure 3-5B). Receptors including the wild-type 
alpha 1, betal and delta subunits with the gamma subunit double mutant also exhibit 
prolonged inhibition while receptors including wild-type alphal, betal and gamma subunits 
with the delta subunit double mutant recover from inhibition within five minutes (data not 
shown). Interestingly, receptors in which three different wild-type subunits are included 
with either the gamma or delta mutant subunit exhibit expression levels similar to wild type 
muscle receptors suggesting that all four subunit types are in fact incorporated into the 
pentameric receptor. These results are consistent with the hypothesis that sequence within 
the intracellular portion of the delta subunit TM2 region (specifically the 5' and 6' residues) 
contributes to the determination of the time-course of recovery from inhibition by bis-TMP- 
10. Moreover, the fact that long-term inhibition requires the presence of at least two 
sensitive subunits (either wild-type delta or gamma(5'_6'delta) mutant) implies that prolonged 
inhibition requires contributions from sequence elements on separate subunits. 



73 

Neuronal Beta Subunit Effects 

Previously published reports have indicated that substitution of a neuronal beta subunit 
for the muscle beta subunit confers sensitivity to long-term inhibition on the resulting 
<x1Pn75 receptor (PN=neuronal beta subunit, either beta 2 or beta4). This result implies that 
sequence elements important for long-term binding are contained, at least in part, within the 
beta subunit sequence. Results from other laboratories have implicated either sequence 
elements in TM2 or the extracellular loop region (ECL) between TM2 and TM3 of muscle 
subunits in regulating the binding of particular noncompetitive inhibitors (see Introduction). 
Thus, these regions are likely candidates for regulation of the binding of bis-TMP-10 as 
well. In order to characterize the mechanism for the selective long-term inhibition of 
neuronal nAChRs, two pairs of chimeric beta subunits were created which exchange eight 
amino acids of either the TM2 or ECL region between muscle (pi) and neuronal beta 
subunits (p4 in this case). Although beta2 and beta4 are identical within this region of 
TM2. the beta4 subunit was chosen for these exchanges because this subunit is more 
prevalent in receptors of the autonomic ganglia and TMP has been demonstrated to be 
effective as a ganglionic blocking agent. Receptors resulting from coexpression of the 
beta4 subunit with alpha3 are likely to represent a reasonable approximation of one class of 
peripheral nAChRs. 

Chimeric DNAs exchanging sequence coding for eight amino acids of the TM2 (in 
underline below) or ECL regions (in double underline below) between a neuronal beta 
subunit (P4) and the muscle beta subunit (pi) were constructed by overlap extension PCR 
(Horton et ai, 1989). The TM2 chimeric region extends from position 4' to position 1 V 
including the position homologous to the inner polar site of Leonard etal. (1988) at which 
the charged amino group of the local anesthetic QX-222 has been hypothesized to bind in 
muscle-type nAChRs (position 6'). The exchanged ECL region begins at position 18' 
incorporating the last three residues of the putative TM2 region and extends through the 
first five residues of the loop between TM2 and TM3. 



74 



intracellular MEMBRANE SPANNING II extracellular 

ALPHA1 MILSISVIiSLTVFLLVTVELIPST 

BETA4 MI LCISVLLAL TFFLL LISKIVPPT 

BETA1 MS LSIFALLTL TVF T ,T ,T ,T .ADKVPET 

4' 11' extracellular 

loop 



These chimeric beta subunits were then expressed with the other muscle subunits (ccl,y,8) 
to produce aipi(p4TM2)y8, aip4(piTM2)y8, aipi(p4ECL)y8 or aip4(piECL)yS receptors. Note 
that only four of the eight amino acids in the underlined region (at positions 4', 6',7' and 
10') differ between the two subunits in the TM2 region while five of the eight amino acids 
exchanged differ between the two subunits in the ECL region. The effects of these 
exchanges were initially evaluated in the muscle receptor because only a single beta subunit 
is included per receptor, while neuronal nAChRs include multiple beta subunits. As noted 
above, the presence of a single neuronal beta subunit in combination with the other muscle 
subunits has been previously shown to be sufficient for achieving long-term inhibition after 
co-application of bis-TMP-10 with ACh (Papke et ai, 1994). 

Coinjection of chimeric or wild type beta subunit RNA with RNA coding for the other 
muscle subunits provides for the expression of functional ccipiy5, cxlp4y8, alpl(p4TM2)y8 
and aip4(piTM2)y5 receptors with activation profiles typical of nAChRs. In order to 
interpret data comparing the magnitude of use-dependent inhibition across receptor 
subtypes, it is necessary to first define a relationship between the experimental 
concentration of agonist applied and the EC50 for each receptor subtype. Although the 
EC50S for each of the receptor subtypes differ somewhat, the Hill coefficients for all of the 
receptor subtypes are in the range of 1-2, typical of nAChRs (not shown). While aipiy5, 
alp4y8 and cclp4(piTM2)y8 receptors show comparable EC50S in the range of 3 to 8 uM, 
alpl(p4TM2)y8 receptors require about a five fold higher concentration of ACh (30 uM) for 
50% activation. Based on the concentration-response studies, the concentration of ACh to 
be used in specific experiments was determined. This concentration ranges between 5 and 



75 



30 uM depending on receptor type or experimental design and is noted in the figure 

legends. 

Dependence of Long-Term Inhibition by Bis-TMP-10 on Sequence in the 
TM2 Region 

The time -course of recovery from inhibition by bis-TMP-10 was examined for a number 
of different subunit combinations (Figures 3-6, 3-7, 3-8). While normal muscle-type 
receptors consistently recover from inhibition within five minutes after co-application of 30 
uM ACh and 2 uM inhibitor, alpl(P4TM2)y5 chimeric receptors show more prolonged 
inhibition (Figure 3-6). In order to demonstrate a reciprocal dependence of this effect, the 
time-course of recovery from inhibition of aip4y6 receptors after co-application of 2 uM 
bis-TMP-10 and 30 uM ACh was compared with that of aip4(piTM2)Y5 receptors (Figure 
3-7). While aip4y8 receptors remain about 73% inhibited after five minutes, 
al(J4(PiTM2)y6 receptors recover to near control levels. Additionally, coexpression of the 
neuronal a3 subunit with the chimeric p4(piTM2) subunit produces receptors which recover 
completely from inhibition within five minutes, while normal a3p4 receptors remain about 
93% inhibited (Figure 3-8). It should also be noted that continuous application of 2 uM 
bis-TMP-10 to either alpl(P4TM2)Y5 or a3P4 receptors for up to one minute in duration 
without co-application of agonist does not produce any significant inhibitory effects 
providing further evidence of the use-dependence of this form if inhibition (data not 
shown). 

The rapid recovery of oc3p4(piTM2) receptors from inhibition by bis-TMP-10 
demonstrates that the neuronal a3 subunit is insensitive to long-term inhibition after 
application of bis-TMP-10. The sensitivities of the neuronal cx2 and a4 subunits to 
inhibition by bis-TMP-10 were also evaluated. While attempts to get expression of 
a4P4(piTM2) receptors were unsuccessful, <x2p4(piTM2) receptors recover completely within 
five minutes from the inhibition elicited with co-application of 30 uM ACh and 2 uM bis- 
TMP-10 (data not shown). Since the a4 and cc2 subunits are identical within the TM2 









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80 

domains, it is hypothesized that these alpha subunits are not sensitive to long-term 
inhibition by bis-TMP-10; however, it is interesting to note that cc7 receptors do exhibit 
long-term inhibition after application of bis-TMP-10. 

For al(3l(p4TM2)Y8 receptors, examination of the relative amount of inhibition remaining 
five minutes after co-application of varying concentrations of bis-TMP-10 with ACh 
indicates that long-term inhibition of this subtype has an IC50 in the range of 30 nM (Figure 
3-9). Previous reports indicate that inhibition of oc3P4 receptors by bis-TMP-10 has an 
IC50 of about 200 nM. It should be noted that, for use-dependent inhibitors, the observed 
IC50 is dependent upon open probability (i.e., agonist concentration). Thus, some 
variability in IC50 across receptor subtype may be attributable to differing levels of open 
probability across experiments. 

In order to examine the rate of recovery from inhibition for individual receptor subtypes, 
ACh was applied at time points beyond five minutes after co-application of ACh with a 
saturating concentration of bis-TMP-10 (2 uM) and residual inhibition was evaluated 
(Figure 3-10). For these experiments, 2 uM bis-TMP-10 alone was applied continuously 
for 15-20 seconds prior to application of ACh in the continued presence of the same 
concentration of bis-TMP-10. The ACh concentrations used were either 10 uM for the 
aipiy5, alp4Y8. alpl(p4TM2)Y8, alp4(PiTM2)v8 subunit combinations or 100 uM ACh for 
oc3P4 receptors. It is possible to estimate a time constant of recovery from inhibition by 
fitting these data with a single exponential. Muscle-type receptors show the most rapid 
recovery from inhibition with a time constant of recovery (x r ) of about 3 minutes, while 
chimeric aipi(P4TM2)Y8 receptors exhibit the most prolonged inhibition (x r =81 minutes). 
As expected, a3p4 receptors also show prolonged inhibition with a time constant of 
recovery of about 70 minutes. The rates of recovery of aip4(piTM2)y8 (x r =6 minutes) and 
a3P4(piTM2) (x r =3 minutes) receptors are most comparable to that of muscle-type receptors, 
while the recovery rate of alp4y8 receptors falls intermediate (x r =16 minutes). 



81 



1.2 



c 1 

o 
o 

2 0.8 

> 

c3 

o 0.6 
c 

0) 

§ 0.4 

a- 

00 

<D 

* 0.2 



alpl(p4TM2)y5 




i 1 1 nil i i i i mil i i_i 





0.0001 0.001 0.01 0.1 1 10 

Concentration (|J,M) 



I I Hill I i i mill | i i ii nil 



100 1000 



Figure 3-9. Concentration dependence of residual inhibition of aipi(P4TM2)y5 
receptors. Each data point represents the mean peak response of at least 4 oocytes 
to 30 uM ACh alone five minutes after coapplication of 30 uM ACh with the 
indicated concentration of bis-TMP-10. Data are expressed relative to the response 
to an initial control application of ACh alone. 



Figure 3-10. Recovery from inhibition by bis-TMP-10 as a function of time. The data 
points at t=0 minutes represent a measure of total inhibition observed after coapplication 
of ACh with 2 uM bis-TMP-10 (see Methods). Data points at t=5, t=10 or t=15 minutes 
represent mean values of residual inhibition measured after application of ACh alone. 
Since for alply8 receptors, full recovery was effectively achieved after only 10 minutes of 
wash, the data at the 15 minute time point for this receptor subtype is omitted for clarity. 
All drug applications are separated by five minute wash periods. The concentration of 
ACh in each experiment is either 10 uM (aipiy8, aipi(P4TM2)y5, aip4y5 and ccip4(piTM2)y8) 
or 100 uM (a3p4 and a3P4(piTM2)). All data points represent the mean responses of at least 
4 oocytes and are fit with an equation describing exponential recovery (see Methods). In 
some cases (e.g., aiply5), the recovery process appears to contain two components. 
However, the normalized responses at later time points reflect the minimal contribution of 
response rundown over time. 



83 






1.0 



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\\ 




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— ■* - alp4y5 

X— alp4(piTM2)y8 

- - -B- - alply5 
# — a3p4(piTM2) 


\\ 


■ i i i ■ i i i i i i i i i i i i i i 


i . . . i 


\\ 

■ i i i i \ i\i i i i i 



2 4 6 8 10 12 14 16 

Time (minutes) 



84 

Effects of Exchange of the ECL Region on Time-Course of Inhibition by 
Bis-TMP-10 

The bis-TMP-10 sensitivity of receptors resulting from the exchange of the extracellular 
loop region was also evaluated (Figure 3-11). Coinjection of the p4(piECL) subunit with 
either the other muscle subunits (al,y,5) or the neuronal alpha3 subunit provides for the 
expression of functional nAChRs. Substitution of the pi ECL sequence does not reverse 
the long-term inhibition normally observed with co-application of bis-TMP-10 and ACh to 
either the aip4y8 or a3p4 subunit combinations. Chimeric <xip4(piECL)y8 receptors have a 
time constant of recovery of about 12 minutes (Figure 3-1 1 A). It should be noted however 
that for aip4(piECL)y8 receptors, response rundown over time is observed so for the 
purposes of calculating a recovery rate, responses at each time point are normalized to the 
amount of rundown observed in response to consecutive applications of ACh alone in 
control oocytes. Similar rundown is observed for a3p4(piECL) receptors. However, for 
this receptor subtype, normalization for rundown can not correct for the observed 
biexponential recovery from inhibition. Thus, data measuring recovery for this receptor 
subtype are plotted together with measurements for rundown and compared to similar 
measures for <x3p4 receptors (Figure 3-1 IB). Even taking rundown into account, only 
slight effects of substitution of the extracellular loop region are observed. The reciprocal 
chimeric beta subunit pi(P4ECU was also tested for bis-TMP-10 sensitivity. Coinjection of 
the pi(P4ECL) subunit with the other muscle subunits (oc,y,S) provides for the expression of 
functional nAChRs. As would also be predicted from the above results, chimeric 
aipi(P4ECL)y5 receptors show little residual inhibition five minutes after application of bis- 
TMP-10 (tr=6.5 minutes). Thus, the capacity to regulate the time course of inhibition by 
bis-TMP-10 appears to be limited to sequence within the N-terminal half of TM2. 



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87 

Mechanism of Inhibition of nAChRs by Bis-TMP-10 

Although the above results are consistent with a role for sequence in the muscle delta 
and neuronal beta subunit TM2 regions in determining the time-course of recovery from 
inhibition by bis-TMP-10, from these data alone, it is not clear whether these effects are 
mediated via a direct contribution of the respective TM2 regions to a binding site for bis- 
TMP-10. It may be the case that sequence elements in TM2 participate in the exposure of a 
bis-TMP-10 binding site distinct from the site of sequence exchanges in TM2. 

Inhibition by Bis-TMP-10 Is Independent of Voltage 

Since the residence time of the previously characterized open-channel blockers QX- 
222 and QX-3 14 has been shown to be dependent on membrane voltage (Leonard et al, 
1988; Neher and Steinbach, 1978) and block by a variety of bis-ammonium compounds 
has also been shown to be voltage-dependent (Ascher et al, 1979; Bertrand et al, 1990; 
Kurenny et al, 1994; Zhorov et al, 1991), we hypothesized that inhibition by bis-TMP-10 
should also show voltage-dependence if bis-TMP-10 is predominantly charged and 
inhibition occurs via binding to the chimeric TM2 region directly. 

The voltage-dependence of bis-TMP-10 inhibition of aipi5 receptors was assessed by 
measuring the response to ACh alone at a holding potential of -50 mV five minutes after co- 
application of ACh with bis-TMP-10 at a holding potential of either -50 mV or +20 mV. 
This response was then compared to an initial response to ACh alone at a holding potential 
of -50 mV. Five minutes after co-application of 2 uM bis-TMP-10 with 30 uM ACh at a 
holding potential +20 mV, the inhibition by bis-TMP-10 is not significantly different from 
that observed after application of bis-TMP-10 with ACh at -50 mV (not shown). Because 
it may be the case that any effects of membrane potential on inhibition can not be detected at 
high concentrations of inhibitor, this experiment was repeated at a bis-TMP-10 
concentration of 500 nM and holding potentials of -80 and +20 mV (Figure 3-12). 



Figure 3-12. Residual inhibition of of a(i5 receptors by bis-TMP-10 does not show a 
measurable voltage-dependence. A) For both sets of traces, the responses at the far left 
and far right are to application of 30 uM ACh alone at a holding potential of -50 mV 
while the middle response is to application of 30 uM ACh with 500 nM bis-TMP-10 at a 
holding potential of either -80 mV (top) or +20 mV (bottom). The voltage steps begin 
about 30 s prior to the start of recording and are maintained for the duration of the middle 
traces. Each response is separated by five minutes. B) Mean data for peak response five 
minutes after coapplication of ACh with bis-TMP-10 are normalized to the initial control 
application of ACh alone. The mean data correspond to the traces at far right in A. 



A 



89 



-80 mV 

ACh with bis-TMP-10 





+20 mV 

ACh with bis-TMP-10 



100 nA 



30 s 



B 



In 



& 



o 0.75 



> 



T = 5 minutes 




-80 mV +20 mV 

Holding potential 



90 

Similar results were observed. Thus, membrane potential does not appear to affect the 
long-term inhibition of aipiS receptors by bis-TMP-10. 

Interestingly, al(3l5 receptors exhibit more inward rectification of current than do either 
muscle or al (31 y receptors (Figure 3-13). Because holding potential does not have any 
measurable effect on long-term inhibition by bis-TMP-10, it also does not appear that the 
process underlying current rectification influences the ability of bis-TMP-10 to bind to the 
channel. 

Since al(3l(p4TM2)y5 receptors resemble neuronal nAChRs in their time-course of 
recovery from inhibition but maintain the linear current-voltage relationship typical of 
muscle-type nAChRs (Figure 3-14, panel B), it is possible to examine the effects of voltage 
on sensitivity to inhibition independent of any potential effects of voltage on channel 
gating. A voltage step to +20 mV for the duration of the co-application of 30 uM ACh with 
2 ^M bis-TMP-10 does not increase the relative magnitude of inhibition of aipi(|J4TM2)y8 
receptors from that observed with a steady holding potential of -50 mV (Figures 3-14 and 
3-16). Thus, for aipi(P4TM2)y8 receptors also, under these conditions at least, the binding 
of bis-TMP-10 to its activation-sensitive site appears to be independent of membrane 
voltage. It should be noted that this experiment examines only the voltage-dependence of 
the onset of inhibition. Therefore, it is possible that some measure of voltage-dependence 
would be observed at a lower concentration of bis-TMP-10. However, the fact that no 
affect on the onset of inhibition was observed even at a positive holding potential coupled 
with the fact that no voltage-dependence of inhibition is observed for a(35 receptors at a 
lower concentration of bis-TMP-10 (500 nM) makes this possibility seem somewhat less 
likely. It may also be possible that the process underlying recovery exhibits a voltage- 
dependence not detected in these experiments. 

The same protocol was used to examine the dependence of inhibition on membrane 
voltage for the a3p4 receptor subtype (Figure 3-15). However, since neuronal receptors 
show pronounced inward rectification (Figure 3-15, panel B), a lack of inhibition at 



91 



A 

al(3ly5 



1.5i 



current 

ftlA) 




1.5 






voltage 

(mVf 



50 



B 



8 



a3p4 



current 

(nA/100) 




8 



voltage 

(mVf 



"50 



c 



1.5 



alply 

current 

(uA) 



-1.5 




D 



6.5i 



al(3l5 



current 

(nA/100) 




voltage 

(mVf 



-6.5* 



50 



Figure 3-13. Representative current-voltage relations for (A) muscle-type, (B) neuronal 
a3P4, (C) a(Jy and (D) a(i8 nAChRs. The holding potential was ramped from -50 mV to 
+50 mV in the plateau phase of the response to a prolonged application of ACh. 
Measurements were made in Ringers solution with barium substitued for calcium. 



Figure 3-14. Long-term inhibition of aipi(P4tTM2)y5 receptors by bis-TMP-10 is 
independent of voltage. A) An initial control application of 30 uM ACh (far left) is 
followed by either successive applicat ons of ACh alone (upper trace) or by a single 
coapplication of ACh with 2 uM bis-TMP-10 followed by subsequent application of ACh 
alone (lower trace). The timing of the voitage step from -50 mV to +20 mV is represented 
by the gray line and begins about 30 s prior to and ends about 30 s after the peak of the 
middle response in each case. Five minute wash periods separate each response. Mean 
data are shown in Figure 3-16. B) A representative current-voltage relationship during the 
plateau phase of the response to extended application of 30 uM ACh alone is shown. The 
mean reversal potential for aipi(P4tTM2)y5 receptors is -4.0±2.1 mV (n=3). Holding 
currents during ramps in the absence of agonist were point to point subtracted. 



A 



1 



93 



oc1(31((34TM2)y5 



r\ 



V 



r 



ACh alone 



120 nAL 



60s 



k- 



AChwithbis-TMP-10 



180nA|_ 



60s 



B 



1.5 1 



Current (|iA) 




■1.5 J 



50 

Voltage (mV) 















Figure 3-15. Long-term inhibition of a3(J4 receptors by bis-TMP-10 is independent of 
voltage. A) An initial control application of 100 uM ACh (far left) is followed by either 
successive applications of ACh alone (upper trace) or by a single coapplication of ACh 
with 2 nM bis-TMP-10 followed by subsequent application of ACh alone (lower trace). 
The timing of the voltage step from -50 mV to +20 mV is represented by the gray line 
and begins about 30 s prior to and ends about 30 s after the peak of the middle response 
in each case. Five minute wash periods separate each response. Mean data are shown in 
Figure 3-16. B) A representative current-voltage relationship during the plateau phase of 
the response to extended application of 100 uM ACh alone is shown. The mean reversal 
potential for oc3p4 receptors is -1 1.0±2.3 mV (n=4). 



95 



A 






oc3(34 










ACh alone 




150 n A 



30 s 




AChwithbis-TMP-10 



300 nA 



30 s 



B 



2 ., 



Current (nA/100) 




-2- 



Voltage (mV) 



ww***"^ 



50 



96 



a 
o 
o 



> 



3 0.5 



u 

1/3 

c 
o 
a. 

c/J 

U 
06 







T=5 minutes 






ACh alone 
(+20 mV) 



ACh 

withbis-TMP-10 
(+20 mV) 




^^ 



ACh 

withbis-TMP-10 

(-50 mV) 



Figure 3-16. Mean normalized data for peak response to ACh at the 5 minute time 
point (corresponding to traces at far right in Figures 3-14A-A and 3-14B-B) are shown. 
Each pair of bars represents the normalized response of either alpl(P4TM2)y5 or a3(J4 
receptors to application of either 30 (alpl(|34TM2)Y5) or 100 uM (a3(34) ACh alone at a 
holding potential of -50mV five minutes after one of three experimental conditions: 
from left to right, application of ACh alone during a voltage step to +20 mV, 
coapplication of ACh with bis-TMP-10 during voltage step to +20 mV or coapplication 
of ACh with bis-TMP-10 at a constant holding potential of -50 mV. 



97 

positive potentials may result from either a voltage-dependence of inhibition itself or a 
voltage-dependence for channel opening. Although neuronal nAChRs pass very little 
outward current at depolarized potentials, co-application of 100 uM ACh with 2 uM bis- 
TMP-10 during a voltage step to +20 mV produces about 75% residual inhibition as 
assessed with application of ACh alone at a holding potential of -50 mV five minutes after 
co-application of inhibitor with ACh (Figure 3-15, lower trace and Figure 3-16). This 
inhibition is clearly independent of the minimal rundown observed with control applications 
of ACh alone (Figure 3-15, upper trace). 

Inhibition of nAChRs by QX-314 

Because bis-TMP-10 appears to be a strictly use-dependent inhibitor, it is of interest to 
compare bis-TMP-10 to other more well characterized use-dependent inhibitors such as the 
open-channel blocker QX-314. As noted in Chapter 1, the mechanism of many open- 
channel blockers has been characterized at the single channel level. Nicotinic AChRs 
normally enter into a period of "bursting" upon the binding of agonist. This burst consists 
of flickering between the agonist bound-open and agonist bound-closed states of the 
channel. Sequential channel blockers bind to the open state of the channel at sites contained 
within the TM2 region and cause protracted bursts such that the mean channel open time 
during a single burst is not reduced. At the single-channel level, an analysis of the channel 
closed times will indicate the introduction of a new component into the closed time 
distribution. However, in a whole-cell response a single channel may bind agonist multiple 
times and pass through many periods of bursting. Therefore, it is not immediately clear 
how application of this drug will affect the kinetics of macroscopic responses. It would be 
predicted that the potential for missed reopenings due to protracted bursts associated with 
blockade will be manifest as a decrease in peak response amplitude and total charge flow 
during the course of a macroscopic response to a co-application of ACh with QX-3 14. In 
addition, it would be predicted that the decay phase of response to a co-application of ACh 



98 

with an open channel blocker would be prolonged as the burst times increase with 
application of the drug. In fact, both of these qualities are observed in responses to the co- 
application of QX-314 and ACh in our system (Figure 3-17). The prolonged falling phase 
of the response is consistent with predictions from single channel observations of inhibition 
by QX-314 and contrasts with observations for inhibition by bis-TMP-10, in which the 
falling phase of the response is very rapid, presumably because of the slow off-rate of bis- 
TMP-10. 

Another potential difference between previously characterized inhibition by open- 
channel blockers and the observations for inhibition by bis-TMP-10 is voltage-dependence. 
In contrast to the observations for the effects of bis-TMP-10 on aipi(|34TM2)y8 receptors, 
QX-314 inhibition of this receptor subtype shows a clear voltage-dependence (Figure 3-18) 
indicating that the QX-314 binding site does indeed lie within the membrane electric field. 

Estimation of the Number of Reopenings in a Macroscopic Response from 
Inhibition by QX-314 

Based on the above observations, it appears that our system can detect the functional 

properties which distinguish QX-314 as a sequential channel blocker. With these 

observations in mind, assuming a strict channel block mechanism for QX-314 which for 

muscle receptors occurs only at a concentration of < 40 uM, it may be possible to make an 

estimate of the number of missed reopenings due to inhibition by QX-3 14. Because single 

channel studies indicate that the predominant effect of a sequential channel blocker is to 

increase burst length, it seems likely that the decrease in macroscopic current associated 

with application of the inhibitor may provide an index for the number of multiple openings 

during a whole-cell response to ACh alone. If inhibition by QX-3 14 is associated with 

missed reopenings due to the prolongation of bursts in the presence of inhibitor, then the 

difference between the integrated net charge of the control response to ACh alone and the 

response to the co-application of ACh with a sequential channel blocker should represent a 

lower limit for the number of multiple openings during a whole-cell response. 






Figure 3-17. Effects of the short-term open channel blocker QX-314 on the responses 
of aipiyS receptors. The gray lines indicate responses in the presence of inhibitor while 
black lines represent control responses to ACh alone. For each set of responses, the 
thick black lines above the traces show the period of drug application. The inset to 
each set of traces displays the same responses scaled to equivalent amplitudes. 



100 



A 



lOO^tMQX-314 



ACh 



1 jliM ACh 




\ ■ mi ———»*— ■■ »>i I i — 



30nA| 



15 s 




B 



100|liMQX-314 



ACh 




3|lMACh / 200 nA 



15s 




c 



100(iMQX-314 



10 |iM ACh 




10 s application of ACh alone 
10 s application of ACh in 
presence of 100 |iM QX-314 



101 












A 



-100 mV 






-80 mV 



-50 mV 






B 



30 ^M ACh alone 

30 nM ACh with 
30 ^M QX-3 14 



In 



Q 
C 
U 



4) 

> 



0.75- 



— 05- 



c 
o 



0.25- 



I 








-1 1 1 1 1 

-100 -80 -60 

Holding Potential (mV) 



-40 



Figure 3-18. Inhibition of al(il(P4TM2)Y8 receptors by QX-314 is voltage-dependent. 
Representative traces of the response to either application of 30 nM ACh alone or 30 ^M 
ACh co-applied with 30 \M QX-3 14 are shown in A. Mean data for the peak response to 
co-application of ACh with QX-3 14 are shown in B. Data are normalized to the response 
to a control application of 30 ^M ACh alone at the indicated potential. 



102 

By calculating the net charge flow in response to a 10 s application of ACh in the presence 

of a high concentration of QX-3 14, it is possible to estimate an upper limit for the number 

of single channel reopenings during a macroscopic response. Using this analysis, it seems 

that, at higher agonist concentrations, response magnitude becomes increasingly dependent 

upon multiple openings (Figure 3-19). Furthermore, it appears that nearly 80% of the 

whole-cell response to 10 p.M ACh results from multiple openings. This value implies that 

responding receptors probably reopen 5 or more times during the time-course of our drug 

applications. 

Preapplication of QX-314 Does Not Protect nAChRs from Long-Term 
Inhibition by Bis-TMP-10 

The protracted response time-course and voltage-dependence of inhibition by QX-314 
and the lack of these effects for bis-TMP- 10 inhibition of nAChRs implies a difference in 
the mechanism of action of these compounds. Specifically, although the beta subunit TM2 
chimeras demonstrate a dependence of inhibition on sequence in the TM2 region, the lack 
of voltage-dependence for this inhibition suggests an indirect effect of TM2 rather than 
binding of the inhibitor directly to this region. It was hypothesized that, if bis-TMP- 10 
binds to the TM2 region directly, a pre-application of QX-314 should protect from long- 
term inhibition. To evaluate this hypothesis for chimeric aipi((54TM2)y5 receptors, we 
applied 100 uM QX-3 14 alone continuously for one minute prior to and throughout a 10 s 
co-application of 2 uM bis-TMP- 10 with 5 ^M ACh until 1 minute after the co-application 
of agonist and long-term inhibitor (Figure 3-20). Residual inhibition was then evaluated 
with applications of ACh alone at 3 and 6 minutes after co-application of ACh with 
inhibitor. While this receptor subtype consistently recovers within 3 minutes from 
inhibition elicited using the same application protocol without the bis-TMP- 10 application 
(middle trace), co-application of bis-TMP- 10 in the presence of QX-314 consistently 
produces long-term inhibition (about 90% at t=3 minutes) that is not significantly different 
from control applications of bis-TMP- 10 with ACh (compare upper and lower traces). 






103 







1 10 1 00 

Concentration ACh (|iM) 



1000 



estimated percentage of blocked 
multiple openings 

-0 peak response to [ACh] 



Figure 3-19. Estimation of missed reopenings due to inhibition by QX-314. Normalized 
peak response calculated as the fraction of maximum response and the approximate 
proportion of current which results from channel reopenings is plotted versus 
concentration of acetylcholine for alply8 muscle-type nAChRs. The percentage of current 
resulting from multiple openings was calulated from the fraction of current inhibited by 
application of 100 uM QX-314 in the presence of varying concentrations of ACh. 



104 



al(3l((54TM2)y5 




B 






100 nA 



30 s 



5 uM ACh 

2uM 
bis-TMP-10 



100 uM 
QX-314 



Figure 3-20. Effects of the presence of QX-314 on long-term inhibition of alpl(|34tTM2)Y5 
receptors by bis-TMP-10. The thick lines above the traces indicate the duration of 
agonist and/or inhibitor application. In each set of three traces, the first trace (from left) 
represents the response to a 10 s application of 5 nM ACh, while the second trace (from 
left) represents the response to either a coapplication of 5 \M ACh with 2 uM bis-TMP- 
10 (A, top), an application of 5 uM ACh in the continued presence of 100 uM QX-314 
(B, middle) or 5 uM ACh coapplied with 2 uM bis-TMP-10 in the continued presence of 
100 uM QX-314 (C, bottom). In all cases, the third trace (from left) represents the 
response to ACh alone applied after a three minute wash period. 



105 

These results demonstrate a lack of effect of QX-314 on inhibition by bis-TMP-10. 
However, because of the potential for multiple intraburst blocking and unblocking events 
during the time-course of agonist application in the presence of the short-term inhibitor, this 
experiment was repeated at higher concentrations of QX-3 14 (up to 500 uM) and at more 
negative potentials. No protection from long-term inhibition was observed with voltage 
steps to -80 mV or applications of 500 uM QX- 3 14 (n=3, data not shown). 

The effects of the monofunctional inhibitor TMP (30 uM) on long-term inhibition of 
odpl(p4TM2)yS receptors were also evaluated using the same application protocol (Figure 3- 
21). TMP is able to produce about 30% protection from long-term inhibition. The results 
of both sets of experiments are summarized in Figure 3-22. 

A similar set of experiments was conducted on the a3p4 receptor subtype. The co- 
application of short- and long-term inhibitors was conducted in the same manner as 
described above. Application of 500 uM QX-3 14 produces a small amount of protection 
(about 18%) from long-term inhibition by bis-TMP-10 (Figure 3-23), while application of 
4 uM TMP with bis-TMP-10 (Figure 3-24) limits long-term inhibition to a level that is not 
significantly different from that observed with application of only the short-term inhibitor 
TMP. The results of both sets of experiments on oc3|}4 receptors are summarized in Figure 
3-25. 

Requirements for Long-Term Inhibition by Bis-TMP-10 

Because it appears that the delta subunit of muscle-type nAChRs is sensitive to the TMP 
moiety, we reasoned that distinct structural elements for regulation of the time-course of 
inhibition might be represented on separate subunits. In order to determine if a requirement 
for contributions by at least two TMP-sensitive subunits for long-term inhibition exists, the 
time-course of recovery from inhibition for receptors containing only a single sensitive 
subunit was examined. aipi(P4TM2)y receptors show no measurable residual inhibition 



106 



aipi(p4TM2)y5 



A 




jr **'" » 00Uf H >*■ n ■>%»■ ■ M U D 




B 





30 s 



5 uM ACh 
bis-TMP-10 



30 uM 

TMP 



Figure 3-21. Effects of the presence of TMP on long-term inhibition of aipi(P4tTM2)y5 
receptors by bis-TMP-10. In each set of three traces, the first trace (from left) represents 
the response to 5 uM ACh, while the second trace (from left) represents the response to 
either an application of 5 ^M ACh in the continued presence of 30 uM TMP (A, upper) or 
a coapplication of 5 uM ACh with 2 |iiM bis-TMP-10 in the continued presence of 30 uM 
TMP (B, lower). In each case, the third trace (from left) represents the response to ACh 
alone applied after a three minute wash period. 



107 



al(3l(p4TM2)y8 



i 



C 

o 
o 

2 

■ — 
— 

13 



aS 

I 

4) 

OS 



0.5 _ 







55 



sS 






•♦ 



• ♦ 



o 



Time (minutes) 



a 


ACh 


♦ bis-TMP-10 


o 


QX-314 


• QX-314 plus bis-TMP-10 


A 


TMP 


A TMP plus bis-TMP-10 



Figure 3-22. The scatter plot displays mean data for the experiments described in Figures 
3-20 and 3-21. The points at t=0 minutes correspond to the second trace (from left) in 
each of the sets of traces in Figures 3-20 and 3-21. The data points at three and six 
minutes show the mean responses to ACh alone after washout of inhibitor and are used as 
measures of recovery from inhibition. Overlapping plot symbols were shifted by about 30 
s to aid in the viewing of all data points. 



108 



cc3(34 




v- 



B 





C 




30 s 



5 |iM ACh 

2u\M — 
bis-TMP-10 



500 uM 
QX-314 



Figure 3-23. Effects of the presence of QX-314 on long-term inhibition of a304 receptors 
by bis-TMP-10. The thick lines above the traces indicate the duration of agonist and/or 
inhibitor application. In each set of three traces, the first trace (from left) represents the 
response to a 10 s application of 100 uM ACh while the second trace (from left) 
represents the response to either a coapplication of 100 uM ACh with 2 uM bis-TMP-10 
(A, upper), an application of 100 uM ACh in the continued presence of 500 uM QX-314 
(B, middle) or 100 uM ACh coapplied with 2 uM bis-TMP-10 in the continued presence 
of 500 uM QX-314 (C, lower). In all cases, the third trace (from left) represents the 
response to an application of 100 pM ACh alone after a 10 minute wash period. 



109 



<x3p4 



A 





30 s 



B 





30 s 



. 5 |IM ACh 
- 2uM = 
bis-TMP-10 



30 uM 

'tmp 



Figure 3-24. Effects of the presence of TMP on long-term inhibition of a3P4 receptors 
by bis-TMP-10. In each set of three traces, the first trace (from left) represents the 
response to 100 \iM ACh, while the second trace (from left) represents the response to 
either an application of 100 uM ACh in the continued presence of 4 uM TMP (A, upper 
traces) or a coapplication of 100 uM ACh with 2 uM bis-TMP-10 in the continued 
presence of 4 uM TMP (B, lower traces). 



110 



1.25 



oc3(34 



1 - 



<u 

C 

o 
fil 
to 
B 

06 

-a 

n 0.5 



o 
Z 








Time (minutes) 



D ACh 


+ bis-TMP-10 


O QX-314 


• QX-314 plus bis-TMP-10 


A TMP 


A TMP plus bis-TMP-10 



Figure 3-25. The scatter plot displays the mean data for the experiments described in 
Figures 3-23 and 3-24. The points at t=0 minutes correspond to the second trace (from 
left) in each of the sets of traces of Figures 3-23 and 3-24. The data points at ten minutes 
show the mean responses to ACh alone after washout of inhibitor and are used as 
measures of recovery from inhibition. Overlapping plot symbols at t=0 minutes were 
shifted in time to aid in the viewing of all data points. 



Ill 

five minutes after co-application of 30 ^M ACh with 2 |iM bis-TMP- 10 (n=6, data not 
shown) indicating that the delta subunit is required for long-term inhibition of receptors 
containing the chimeric pi(|34TM2) subunit. 
Long-Term Inhibition Is Dependent upon Compound Length 

Collectively, these data seem to suggest that binding of bis-TMP- 10 is regulated by 
distinct sequence elements in TM2 contributed by separate subunits. Based on the voltage- 
independence of inhibition and the relative lack of effect of application of QX-3 14 on 
inhibition, it does not appear that bis-TMP- 10 is binding to the portion of TM2 influenced 
by the membrane electric field or accessible to open channel blockers. If binding of both 
TMP moieties of the bi-functional compounds is necessary for long-term inhibition, we 
reasoned that the distance between TMP moieties might represent a constraint on inhibitory 
activity and furthermore, give some indication of the level of bis-TMP- 10 binding within 
the receptor complex. To test this hypothesis, the amount of inhibition remaining at time 
points 5 and 10 minutes after co-application of inhibitor with ACh to either chimeric 
al(3l(P4TM2)y5 or neuronal a3p4 receptors was assessed for bi-functional TMP molecules 
differing only in the length of their carbon linker. TMP, bis-TMP-4, bis-TMP-6, bis- 
TMP-8, bis-TMP- 10 and bis-TMP- 12 were tested for their inhibitory effects (Figure 3-26). 
For both receptor subtypes, all of the compounds including the monofunctional inhibitor 
TMP show some inhibitory activity at the time of co-application with ACh. However, no 
significant residual inhibition of alpl(P4TM2)y8 receptors results from co-application with 
ACh of compounds with linkers of eight carbons or less while pronounced residual 
inhibition is present after application of inhibitors with linkers of ten or more carbons (top 
panel) . In contrast, for neuronal nAChRs (a3p4), all bi-functional compounds show some 
degree of residual inhibitory activity at time points 5 and 10 minutes after the time of co- 
application with ACh and the magnitude of residual inhibition increases with increasing 
compound length (bottom panel). Similar results were observed for inhibition of the 
neuronal a4p2 subunit combination by the bis-TMP-n compounds (not shown). 






112 



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113 

Evaluation of Effects of Intracellular^ Applied Bis-TMP-10 

Data from two-electrode voltage clamp studies are consistent with bis-TMP-10 
mediating its effects via sequence elements available on the extracellular face of the 
receptor. This hypothesis was tested directly by applying a saturating concentration of bis- 
TMP-10 to the inside of the membrane and testing for effects on nAChR responses. The 
cut-open oocyte vaseline-gap voltage clamp preparation allows exchange of both the 
intracellular and extracellular solutions (Costa et al, 1994). A perfusion pipette which 
penetrates the oocyte from below controls the intracellular solution (100 mM KC1 in most 
cases) and acts as voltage sense pipette. The interior of the cell is voltage clamped to 
ground while the exterior of the oocyte is bathed in Ringers solution and clamped to a 
command potential (+50 mV relative to the interior in this case for a holding potential of -50 
mV). Vaseline seals between the oocyte and the recording chamber separate the internal and 
external compartments. Therefore, it is possible to test the effects of bis-TMP-10 applied 
to the intracellular side of the membrane independent of extracellular effects. 

The time course of exchange of the intracellular solution was assessed by switching 
from a 100 mM KC1 internal solution to an N-methyl-D-glucamine (NMDG) containing 
solution of equivalent osmolarity and measuring the reduction in outward currents of 
alpiy8 receptors in response to repeated applications of ACh over time (Figure 3-27). 
Initial observations for the exchange of a colored dye in the internal solution indicated that 
the time necessary for solution exchange within the pipette was about 10 minutes with the 
syringe pump set to 0.01 ml/hr. With this result in mind, measurements were made 
starting at 15 minutes after the switch to the NMDG-based solution. Outward currents are 
reduced by about 60% at the 15 minute time point compared with no reduction the response 
of control oocytes at the same time point perfused continuously with 100 mM KC1. By 24 
minutes after the switch to the NMDG-based internal solution, outward currents of muscle 
nAChRs are reduced by 90% without a detectable reduction in the responses of control 
oocytes. Thus, solution exchange should be complete within 25 minutes. 









Figure 3-27. Substitution of N-methyl-D-glucamine shows the time-course of exchange 
of solution for internal perfusion in cut-open oocyte vaseline-gap voltage clamp 
technique. A) The responses of ccipiyS injected oocytes to an application of 10 uM ACh at 
a holding potential of +50 mV are plotted relative to a control response measured 
immediately prior to either continued perfusion of a solution containing 100 mM KC1 
(230 mOsm, pH 7.3) or activation of a second syringe pump containing a solution of 130 
mM N-methyl-D-glucamine (ph 7.3, 230 mOsm). Based on observations of exchange of 
a colored dye, the first measurements were made 15 minutes after switching to the second 
pump. Internal perfusion rate was 0.1 mL/hr. B) Representative traces of responses to 10 
uM ACh at a holding potential of +50 mV in either KC1 (gray) or NMDG-based (black) 
internal solutions. The response in the presence of internal NMDG ocurrs 24 minutes 
after activation the control response. 



115 



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IS 



T 

■ 

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■ 1 30 mM NMDG internal 



T 

■ 

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18 



—r 
21 



Time (minutes) 



T 

■ 
i 



24 



— I 
27 



+50 mV 




-50 mV 



116 

The effects of intracellular^ applied bis-TMP-10 were evaluated by perfusion of a KC1- 
based internal solution containing 2 uM bis-TMP-10 and measuring responses to 5 s 
applications of 10 uM ACh in 3 minute intervals beginning 15 minutes after the start of 
internal solution exchange. For both aipi(P4TM2)y8 and a3p4 receptors, continuous 
perfusion of bis-TMP-10 to the internal face of the membrane in this manner has little if any 
effect on inward currents in response to application of ACh (Figure 3-28). Consistent with 
results from two-electrode voltage clamp experiments, application of 2 uM bis-TMP-10 
with 10 uM ACh to the external face of the membrane causes inhibition of nAChRs (alpy8 
response shown in figure). However, repeated application of ACh in the presence of 
internal bis-TMP-10 does not cause any significant inhibition of inward currents at a 
holding potential of -50 mV (Figure 3-28B). Consistent with the lack of any voltage- 
dependence for inhibition by bis-TMP-10 applied to the external face of the membrane in 
two-electrode voltage clamp studies, voltage steps to positive potentials do not affect the 
lack of inhibition by internally applied bis-TMP-10 for either aipi((J4TM2)Y6 or a3p4 
receptors (data not shown). 

Inhibition by an Amphipathic Analogue of Bis-TMP-10 

Assessing the significance of the requirement for a long linker for long-term inhibition 
of aipi(P4TM2)y6 receptors requires knowledge of the relative contributions of the 
piperidinol moieties versus the aliphatic linker chain to the long-term binding of bis-TMP-n 
compounds. For example, the requirement for a 10 carbon distance between TMP moieties 
may reflect the distance between two independent TMP binding sites or may simply reflect 
the strength of hydrophobic interactions between the receptor and the aliphatic chain. The 
fact that TMP is a potent short-term inhibitor of neuronal nAChRs is consistent with the 
hypothesis that the TMP moieties of bis-TMP-10 mediate the rate-limiting interactions for 
determination of the off-rate of bis-TMP-10 from the receptor. However, the relative 
insensitivity of muscle-type nAChRs to even short-term inhibition by the monofunctional 



Figure 3-28. Inhibition of nAChRS by bis-TMP-10 occurrs only when applied to the 
external face of the membrane. A) Representative responses of alplyS receptors to either 
an application of 30 uM ACh alone at a holding potential of -50 mV (gray line) or a 
coapplication of 30 uM ACh with 2 uM bis-TMP-10 (black line) to the external face of 
the membrane. B) Responses of either a3p4 (circles) or aipi(p4TM2)v6 receptors (squares) 
to successive applications of ACh alone to the external face of the membrane in the 
presence of either an internal solution containing 100 mM KC1 only (open symbols) or an 
internal solution containing 100 mM KC1 with 2 uM bis-TMP-10 (closed symbols). The 
first time point shown is 15 minues after an initial control application of ACh alone to 
which the data are normalized. The ACh concentrations used are 100 mM (oc3|34) and 30 
uM (alpl(P4TM2)y5). Some data points are shifted slightly in time to aid in the viewing of 
all data. 












118 



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119 

inhibitor TMP may suggest that the aliphatic chain connecting the TMP moieties of bis- 
TMP-10 contribute to determination of the time-course of recovery aipi(P4TM2)y5 receptors 
from inhibition. In an effort to determine which part of the bis-TMP-10 molecule mediates 
the interactions responsible for long-term inhibition, an amphipathic compound consisting 
of a piperidinol ring linked to a ten carbon aliphatic chain was synthesized (ATMP-10 in 
Figure 1-1). If binding of bis-TMP-10 was strictly mediated by the piperidinol moieties, it 
would be predicted that subunit combinations which are sensitive to long-term inhibition by 
bis-TMP-10 would be insensitive to long-term inhibition by the amphipathic compound 
containing only a single piperidinol ring. In contrast, if chain length dependent inhibition is 
regulated by binding of the aliphatic chain directly, it would be predicted that 
monofunctional TMP compounds coupled to an aliphatic chain of sufficient length would 
produce long-term inhibition while those with shorter length aliphatic chains would 
produce lesser amounts of residual inhibition. Interestingly, it appears that ATMP-10 can 
produce long-term inhibition of both chimeric aipi(P4TM2)y5 receptors and a3(34 receptors. 
However, this inhibition appears be qualitatively different from inhibition by bis-TMP-10. 
Specifically, higher concentrations of ATMP-10 are required to produce maximal inhibition 
(Figure 3-29). For al(Jl((J4TM2)Y8 receptors, the IC50 for residual inhibition by ATMP-10 
five minutes after the co-application of ATMP-10 with ACh is about 1 uM. For a3p4 
receptors, the IC50 for residual inhibition measured in the same manner is about 22 uM. 
These values are about 30-fold and 100-fold greater respectively than the same measures 
for inhibition of these receptor subtypes by bis-TMP- 10. 

Inhibition by ATMP-10 Independent of Activation by Agonist 

In the absence of agonist, a one minute application of a concentration of inhibitor that 
appears to be saturating when applied for 10 s in the presence of agonist, produces 
significant inhibition of both al(Ji((i4TM2)Y5 and a3(i4 receptors five minutes after 
termination of the inhibitor application. For al(3l(p4TM2)y5 receptors, comparable amounts 



120 






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Figure 3-29. Residual inhibition of alpl(p4TM2)y5 and a3(J4 receptors five minutes after 
co-application of varying concnetrations of ATMP-10 with ACh. Each data point 
represents the mean ±SEM of at least 4 responses to each drug application. The 
concentration of ACh used in each case is 30 uM for aipi(p4TM2)y8 receptors and 100 ^M 
for a3p4 receptors. 



121 

of inhibition five minutes after application of inhibitor with or without agonist are observed 
(Figure 3-30). By contrast, for a3(34 receptors, this agonist independent form of inhibition 
is less apparent five minutes after termination of the inhibitor application but continues to 
accumulate with repeated applications of ACh alone at later time points (Figure 3-31). For 
inhibition in the presence of agonist, it is possible to estimate a time constant of recovery 
from inhibition of about 19 minutes for aipi((34TM2)y6 receptors compared to about 34 
minutes for a3|34 receptors. It should be noted however that the presence of a use- 
independent component to inhibition by ATMP-10 complicates measures of recovery rate 
from use-dependent inhibition. Specifically, it is not possible to accurately measure 
inhibition at the time of co-application of agonist with inhibitor in the same manner used in 
the case of inhibition by bis-TMP-10. For inhibition by ATMP-10, a prolonged 
preapplication of inhibitor alone (as was done for measurement of time course of recovery 
from inhibition by bis-TMP-10) would cause a form of agonist independent inhibition 
which is mechanistically and possibly structurally distinct from inhibition requiring the 
presence of agonist. Therefore, for this inhibitor, recovery rate is estimated without data 
for response at the time of co-application of agonist with inhibitor (t=0) and therefore 
should not be viewed as absolutely quantitative. 

Voltage-Dependence of Inhibition by ATMP-10 

As noted above. bis-TMP-10 inhibition of all receptor subtypes tested appears to be 
independent of holding potential. By contrast, inhibition of both aipi(P4TM2)Y5 receptors 
and oGf}4 receptors by ATMP-10 does show a significant voltage-dependence (Figure 3- 
32). For aipi(P4TM2)y8 receptors, co-application of 1 uM ATMP-10 with 30 uM ACh at a 
holding potential of -80 mV produces over 60% residual inhibition of responses to ACh 
alone five minutes later while co-application of agonist with inhibitor at a holding potential 
of +20 mV produces only 35% residual inhibition of responses to ACh alone at the five 
minute time point. For a3|}4 receptors, co-application of 10 yM ATMP-10 with 100 uM 



122 








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ACh at a holding potential of -80 mV produces about 75% inhibition of responses to ACh 
alone five minutes after the co-application of agonist with inhibitor while co-application of 
agonist with inhibitor at a holding potential of +20 mV produces only about 40% residual 
inhibition at the same time point. 
TMP Protects alpi(P4TM2)y5 Receptors from Inhibition by ATMP-10 

It appears that although ATMP-10 behaves as a long-term inhibitor of nAChRs, the 
inhibition by this compound is distinct from that of bis-TMP-10 in its relative potency, 
modes of action and voltage-dependence. It may be the case that these mechanistic 
differences reflect interactions with sites independent of those important for the binding of 
bis-TMP-10. The monofunctional inhibitor TMP does not completely protect 
alpl(P4TM2)y5 receptors from long-term inhibition by bis-TMP-10. In contrast, 
preapplication of 30 uM TMP reduces the inhibition observed five minutes after co- 
application of 2 uM ATMP- 10 with 5 uM ACh from 67±01 % to 27±02% (n=4, data not 
shown). 



CHAPTER 4 
DISCUSSION 



A great deal of research has employed the use of noncompetitive inhibitors to explore 
the relationship between structure and function in ion channels. The muscle-type/Torpedo 
nAChR is the prototype system for these studies both because of its ready availability in 
purifiable quantities and the rigorous characterization of its functional role at the 
neuromuscular junction. The main goal of the work in this thesis is to understand the 
structural basis for the selectivity of bis-TMP-10 for long-term inhibition of neuronal 
nAChRs. By examining the mechanism of inhibition of a class of drugs which show 
selectivity for the long-term inhibition of neuronal nAChRs, these studies attempt to extend 
the analysis of structure-function relationships to neuronal nAChRs. Towards describing 
the structural elements underlying this sensitivity, mutant subunits which exchange 
sequence between the TM2 regions of the delta and gamma muscle subunits, chimeric 
subunits which exchange sequence between the TM2 regions of muscle and neuronal beta 
subunits and chimeric subunits which exchange sequence between the ECL regions of 
muscle and neuronal beta subunits were synthesized and characterized. Analysis of these 
mutants has indicated that the sensitivity to long-term inhibition by bis-TMP-10 is regulated 
by sequence in the TM2 domain. Furthermore, it appears that the presence of at least two 
bis-TMP-10 "sensitive" subunits is required for the induction of long-term inhibition. 
Interestingly however, these data indicate that long-term inhibition by bis-TMP-10 is 
strictly use-dependent within the time frame of application of inhibitor used in these studies 
but apparently not associated with direct binding to site(s) situated within a portion of the 
ion channel pore influenced by the membrane electric field or accessible to open channel 
blockers. The most parsimonious interpretation of these data allows for interactions, upon 

127 



128 

channel activation, between residues located within the TM2 region of neuronal beta 
subunits and sequence elements situated outside of the membrane electric field. It is 
possible that these interactions, in turn, result in the exposure of the use-dependent binding 
site(s)forbis-TMP-10. 

The Mechanism of Action of Mecamylamine Is Distinct from that of Bis- 
TMP-10 

These studies build on previous studies examining sensitivity of muscle nAChRs to 
noncompetitive inhibition by describing a form of voltage-independent long-term inhibition 
of muscle-related aps nAChRs which apparently is mediated by delta subunit sequence 
homologous to a site for which only voltage-dependent inhibition has been previously 
characterized. Because both bis-TMP-10 and mecamylamine show selectivity for the 
inhibition of neuronal and ocP8 nAChRs, it is of interest to contrast the quality of the 
inhibition by these two compounds. Although aps receptors exhibit prolonged inhibition 
after application of either mecamylamine or bis-TMP-10, the inhibition of ap8 and apy 
receptors by mecamylamine appears qualitatively different from the inhibition by bis-TMP- 
10. Specifically, inhibition of muscle-related receptors by mecamylamine does not show 
the same decrease in time to peak response as is the case for inhibition by bis-TMP-10. It 
may be the case that this difference in response waveform reflects a faster off-rate for 
mecamylamine compared to bis-TMP-10; however, ap5 receptors show more residual 
inhibition five minutes after application of mecamylamine than do a3p4 receptors for which 
a clear reduction in time to peak was observed. Thus, it is most likely the case that the 
difference in response waveform reflects a difference in mechanism of inhibition. It is 
possible that mecamylamine has some competitive or non use-dependent effects at aP5 
receptors. As noted in Chapter 1, the literature is in some disagreement on this point. 
Previous studies of muscle nAChR indicate that mecamylamine acts as a noncompetitive 
antagonist (Varanda, etai, 1985) while other studies show mecamylamine to be 






129 

competitive at neuronal nAChRs (Ascher, et al, 1979). Thus, it seems likely that 
mecamylamine can act via both mechanisms. For muscle nAChR, it may be the case that 
competitive or non-use dependent effects with a longer time-course are associated with the 
delta subunit while shorter time-course use-dependent effects are associated primarily with 
the gamma subunit. The mixed effects of mecamylamine may obscure our ability to 
conclude mechanism of inhibition from qualitative inspection of the waveform. 

The inhibition of neuronal a3p4 receptors by mecamylamine is qualitatively similar to the 
inhibition by bis-TMP-10 and in this case does seem to be use-dependent. Furthermore, 
recent data from this laboratory demonstrate that sensitivity to inhibition by mecamylamine 
for oc3p4 receptors is dependent upon the same stretch of sequence in the beta subunit TM2 
region which regulates sensitivity to long-term inhibition by bis-TMP-10 (Webster et al, 
unpublished observations). In contrast to inhibition by bis-TMP-10, inhibition by 
mecamylamine is voltage-dependent with an approximate IC50 of 100 nM for acute 
inhibition and 1 \iM for residual inhibition remaining five minutes after application of 
inhibitor. 

TM2 Determines Kinetics of Long-Term Inhibition by Bis-TMP-10 

Omission of gamma subunit RNA has an effect on the receptor's sensitivity to inhibition 
by bis-TMP-10 that is similar to previously reported effects for substitution of a neuronal 
beta subunit (p4 or |32) for the muscle beta subunit (pi). This result is consistent with the 
hypothesis that neuronal beta subunits and muscle delta subunits share a common structural 
motif which provides a site for the binding of bis-TMP-10 and which is not present in the 
gamma subunit. Exchange of residues at the 5' and 6' positions of TM2 between the 
gamma and delta subunits alters sensitivity to long-term inhibition by bis-TMP-10 
indicating that this site can regulate the binding of bis-TMP-10 to the delta subunit. 
However, aPy^-.^ receptors recover from inhibition by bis-TMP-10 with a time constant 
of 10 minutes whereas ap5 receptors recover from inhibition by bis-TMP-10 with a time 



130 

constant of 25 minutes. The faster recovery rate of the mutant receptors suggests that, 
although the delta subunit 5' and 6' residues are important for regulating sensitivity to long- 
term inhibition by bis-TMP-10, the amino acids at these positions are not the sole 
determinants of time course of recovery from inhibition by bis-TMP-10. Alternatively, it 
may be the case that, because the EC50 for activation of this receptor subtype by ACh is 
high compared with the EC50 for activation of either ap5 or afty receptors, the faster 
recovery rate in part may reflect insufficient activation at the time of co-application of ACh 
with inhibitor to achieve complete inhibition. 

Studies examining the time-course of inhibition of receptors containing chimeric beta 
subunits localize the major structural determinant of sensitivity to long-term-inhibition to an 
eight amino acid stretch in the N-terminal portion of TM2 of neuronal beta subunits. All 
receptor subtypes tested which incorporate the neuronal beta subunit TM2 region paired 
with either a second neuronal beta subunit (neuronal nAChRs) or a delta subunit (neuronal- 
muscle chimeras) exhibit a significant degree of residual inhibition as measured at time 
points five minutes or more after the co-application of agonist with inhibitor. The reversal 
of long-term inhibition upon substitution of the pi subunit TM2 region for the p4 subunit 
TM2 region in alp4(PiTM2)y5 and a3p4(piTM2) receptors in conjunction with the 
observation that substitution of the (31 ECL region does not reverse long-term inhibition 
demonstrates this effect to be specific for the N-terminal portion of TM2. Because long- 
term inhibition of receptors incorporating the (3l((34TM2) subunit also requires the presence 
of the delta subunit, it appears that the TM2 regions of each subunit are required to produce 
long-term inhibition. 

Moreover, if the time-course of recovery from inhibition reflects the time-course of 
dissociation of the TMP moieties of bis-TMP-10 from inhibitory binding sites, the more 
prolonged time-course of recovery of aipi((34TM2)y5 and a3P4 receptors from inhibition by 
bis-TMP-10 compared with the more rapid recovery of aps receptors from inhibition by 
bis-TMP-10 may indicate that the higher affinity TMP interaction is associated with the beta 



131 

subunit. Thus, this interaction would be rate-limiting for the recovery of al(3l(P4TM2)y5 
receptors from inhibition by bis-TMP-10. 

While aip4y8 receptors exhibit prolonged inhibition compared to al(i4(piTM2)Y5 or 
muscle-type receptors, they recover from inhibition more rapidly than the oclpl(P4TM2)Y8 
receptor subtype. This observation perhaps suggests that, when associated with the other 
muscle subunits in alp4y5 receptors, regions of the p4 subunit not contained within the 
TM2 domain may affect the binding of inhibitor directly or otherwise allow for increased 
recovery rate. The (34 subunit has been shown to confer the property of prolonged burst 
kinetics upon the neuronal subunits with which it is expressed (Papke and Heinemann, 
1991). It may be the case that activation properties such as burst duration or channel open 
time which may be specific to individual receptor subtypes can influence recovery rate or 
the probability of block as a function of peak current. However, it seems more likely that 
sequence within the bis-TMP-10 binding site itself influences the time-course of recovery 
from inhibition. It may be the case that, in this subunit configuration, insertion of the beta4 
TM2 sequence into betal provides for exposure of a bis-TMP-10 binding site with a higher 
affinity than that which is present on wild-type beta4. 

Significance of Voltage-Independent Inhibition 

In contrast to studies on the open channel blockers QX-222 and QX-3 14 (Neher and 
Steinbach, 1978), and studies on the symmetrical bis-ammonium series of ganglionic 
blockers (Ascher et al., 1979) in which block has been shown to be dependent on 
membrane potential. bis-TMP-10 inhibition of the receptor subtypes tested in these studies 
seems to be independent of voltage. The lack of voltage-dependence for inhibition brings 
to mind two possibilities: either the noncompetitive binding site is outside of the membrane 
electric field or bis-TMP-10 is uncharged at physiological pH. Although direct evaluation 
of the pKa of bis-TMP-10 has not been possible because of solubility limitations, it is 
known that the monofunctional inhibitor TMP has a pKa in the range of 10-1 1 (Perrin, 



132 

1965). The pKa of simple bis-amino compounds are reduced if the amines are separated 
by short (2-4) carbon chains. However, when the amines are separated by a longer (e.g. 
8) carbon chain, the pKa values of the two ionizable groups both approach that of the 
monofunctional amine. Therefore, with the pKa of the monofunctional piperidine in the 
range of 10-1 1, it is unlikely that the lower of the 2 pKa values of the bis-compound would 
be under 9-10. Thus, at physiological pH both functional groups should be predominately 
charged. 

The voltage independent long-term inhibition of a3|34 receptors by bis-TMP-10 is 
particularly intriguing. As shown in Figure 3-15, the inhibition of a3p4 receptors observed 
five minutes after the co-application of agonist with inhibitor at +20 mV (about 75%) is 
slightly less than that typically observed five minutes after co-application of ACh with bis- 
TMP-10 at -50 mV (about 90%). This effect may represent a voltage-dependent 
component of long-term inhibition but more likely represents an effect of the voltage- 
dependence of channel gating independent of any voltage-dependence for inhibition. In 
either case, at a holding potential of +20 mV no outward current was measured in response 
to control applications of ACh alone, implying that the activation-dependent state associated 
with inhibition is maintained even at positive potentials for which there is no measurable 
outward current. As is the case for some subtypes of ionotropic glutamate receptors and 
certain classes of potassium channels, the inward rectification of neuronal nAChRs has 
recently been shown to be due, in large part, to block by intracellular polyamines at positive 
potentials. Our data suggest that an activated conformation is present at depolarized 
potentials and are consistent with the observation that blockade by intracellular factors 
underlies the process of rectification. Thus, bis-TMP-10 appears to be able to produce 
long-term inhibition of neuronal nAChRs which are gated (or activated) but non-conducting 
as a result of block from the intracellular side. 

A similar situation exists for the inhibition of apS receptors by bis-TMP-10. Although 
this receptor subtype shows pronounced inward rectification (Figure 3-13), there is no 



133 

detectable difference between the inhibition observed for application of 500 nM bis-TMP- 
10 at a holding potential of -80 mV and that observed after application of bis-TMP-10 at a 
holding potential of +20 mV (Figure 3-12). Again, because use-dependent inhibition 
requires prior activation of the channel, this result implies that the process underlying 
rectification is distinct from the process underlying deactivation of the receptor. 

These data cannot rule out the alternative possibility that bis-TMP-10 inhibition is 
mediated via the uncharged species of the inhibitor. Because pKa represents an estimate of 
the equilibrium between uncharged and charged species, if the pKa of the respective 
ionizable groups of bis-TMP-10 is about 10, at pH 7, the concentration of the uncharged 
species would be estimated to be about 1000 times less than the concentration of the 
charged species. 

Inhibition by Bis-TMP-10 Is Distinct from Inhibition by QX-314 

The response waveforms of alplyS receptors in the presence of QX-3 14 show 
profound differences from the responses of alply5 receptors in the presence of bis-TMP- 
10. Specifically, application of QX-3 14 prolongs the falling phase of the response whereas 
inhibition by bis-TMP-10 is characterized by a very rapid decrease in response amplitude. 
This difference is most likely attributable to a difference in the off-rate of the two inhibitors. 
In fact, in the case of bis-TMP-10 inhibition of muscle receptors, bis-TMP-10 may not be 
acting as a sequential channel blocker at all. Although the rapid falling phase of the 
response indicates prolonged inhibition, the time constant of recovery from inhibition for 
muscle receptors is about 3 minutes consistent with relatively short-lived inhibition. 
Therefore, it may be the case that the unbinding of bis-TMP-10 is not state dependent. In 
fact, recent single channel observations by other investigators in this laboratory support the 
hypothesis that although inhibition by bis-TMP-10 requires prior activation of the channel, 
unbinding occurs only after ACh has dissociated from the agonist binding site and thus no 
subsequent activation of the receptor by agonist is observed (Kabakov and Papke, 



134 

unpublished observations). With this idea in mind, it may be the case that, for all subtypes 
of nAChR, inhibition by bis-TMP-10 is mediated by stabilization of an intermediate closed 
state rather than a blocked state. Thus, recovery from inhibition would depend upon the 
unbinding of inhibitor and subsequent conformational change to the resting closed state. It 
is also interesting to note that even receptors incorporating only single "sensitive" subunit 
(e.g., wild-type muscle) or no "sensitive" subunits (e.g., a(3y) are inhibited by bis-TMP-10 
at the time of co-application of ACh with inhibitor. It may be the case that the presence of 
two "sensitive" subunits is required not for induction of inhibition itself, but for the 
stabilization of an intermediate closed state associated only with long-term inhibition. 

Bis-TMP-10 and QX-314 Do Not Compete for the Same Site 

The hypothesis that bis-TMP-10 can produce long-term inhibition of nAChRs which are 
non-conducting as a result of blockade from the extracellular side was evaluated by 
preapplication of the open-channel blocker QX-3 14. This drug was chosen for these 
experiments based upon three criteria: the QX-314 binding site is believed to be located 
approximately 3/4 of the way across the membrane electric field and thus inhibition by this 
compound is voltage-dependent (Neher and Steinbach, 1978); QX-3 14 has been shown to 
interact with residues at homologous positions to those contained within the region of our 
beta subunit TM2 chimeras (Pascual and Karlin, 1997); and QX-314 has a longer residence 
time in the pore than the structurally related local anesthetic QX-222 (Neher and Steinbach, 
1978). Moreover, the inhibition of chimeric al|M(p4TM2)Y8 receptors by QX-314 is 
voltage-dependent indicating that for this subunit combination also, the QX-314 binding 
site is located within the membrane electric field (Figure 3-18). 

For aipi(p4TM2)y5 receptors, the lack of an effect of even very high concentrations of 
QX-3 14 (500 |iM) on the magnitude of residual inhibition after co-application of bis-TMP- 
10 with ACh is consistent with the observed voltage-independence of inhibition by bis 
TMP-10. For oc3[}4 receptors, QX-3 14 does produce a detectable reduction in the 



135 

magnitude of residual inhibition (Figure 3-23). This observation suggests that, in this 
subunit combination, the sites of action for bis-TMP-10 and QX-314 are not totally 
independent. From these data, it is difficult to ascertain whether the partial protection 
observed represents interactions of both drugs at a single site or alternatively, an allosteric 
interaction between two distinct sites, such that the binding of QX-314 decreases the gating 
dependent changes at the site of TMP binding. 

For both receptor subtypes, the short-term inhibitor TMP produces a significantly 
greater degree of protection from long-term inhibition than was observed with QX-314. 
This finding demonstrates a degree of overlap in the sites of action between the mono- and 
bi-functional compounds. The lack of complete protection may suggest a contribution of 
the hydrophobic linker region in stabilizing bis-TMP-10 binding or may indicate that a 
different subset of binding sites are available to the smaller TMP compound. The latter 
hypothesis is supported by the observation of a slight voltage-dependence for inhibition by 
TMP(Papke<?ra/., 1994). 

In the case of a[55 receptors, long-term inhibition by bis-TMP-10 is shown to 
dependent upon the 5' and 6' residues. Similarly, one of the nonidentical residues between 
muscle and neuronal beta subunits in the beta subunit TM2 chimeras is the 6' residue. 
Studies by other investigators have demonstrated sequence at the 6' position to be 
important for the voltage-dependent binding of QX-222 and QX-3 14. Although it may be 
expected that very high affinity binding may not show an appreciable voltage-dependence, 
the voltage-independence of inhibition by bis-TMP-10 coupled with the inability of QX- 
314 to protect from inhibition implies that the site of bis-TMP-10 binding may not be 
contained within the region of exchanged sequence. It may be the case that sequence 
within the TM2 region provides for exposure of potential binding sites to bis-TMP-10 
which are located outside of the membrane electric field. In this case, the sensitivity to 
long-term inhibition may be determined by sequence in the TM2 region (which mediates 
exposure of the inhibitor binding site), while the time course of recovery from inhibition 



136 

would be determined by sequence composing the inhibitor binding site itself. Interestingly, 
all of the subtypes sensitive to long-term inhibition have a polar residue at the 6' position 
and a nonpolar residue at the 10' position suggesting that these residues may be critical for 
the transitions to a bis-TMP-10 sensitive state upon activation of the channel. Because 
long-term inhibition is dependent upon sequence in TM2 for a(58, beta subunit TM2 
chimeric muscle and TM2 chimeric neuronal receptors, it seems reasonable that the 
inhibitor binding site may represent a structural motif which is conserved across subunits. 
For aPY(5'.6'5) receptors, exchange of the 5' and 6' residues with delta could not fully 
account for the prolonged time-course of inhibition observed for aP5 receptors. Thus, it 
may be the case that insertion of these residues provides for the exposure of a bis-TMP-10 
binding site on gamma which has a lower affinity than the one available on the delta 
subunit. For the beta subunit of muscle-type nAChRs, it has been shown that channel 
activation alters the accessibility of substituted cysteines at positions immediately 
extracellular to the region of our chimera (Zhang and Karlin, 1996) and also in the N- 
terminal third of the beta subunit TM1 (Zhang and Karlin 1997). It may be the case that 
these residues contribute to a binding site for bis-TMP-10. 

Because the sensitivity of aipi((i4TM2)Y5 receptors to long-term inhibition by bis-TMP- 
10 requires the presence of both the chimeric beta subunit and the delta subunit, it seems 
that sequence on both of these subunits contributes to bis-TMP-10 binding. It may be the 
case that each sensitive subunit provides a single site for binding independent of the other 
subunits in the pentamer and that two such sites are required for long-term inhibition. 
Alternatively, it may be the case that the coordinated contributions of the delta and chimeric 
beta subunit are required for expression of a single high affinity site. 









137 

Significance of Compound Length Requirement for Long-Term Inhibition 

The results of both the voltage-dependence and protection experiments localize the 
majority of bis-TMP- 10 effects to sites lying outside the membrane electric field. In 
addition, the lack of effect of intracellularly applied bis-TMP- 10 argues for a site accessible 
only from the external face of the membrane. Given the data, it seems logical to speculate 
that these sites lie on the extracellular portion of the receptor, possibly as a part of the 
protein domains forming the extracellular vestibule. In its most extended conformation, 
bis-TMP- 10 is estimated to have a length of between 20 and 23 A. This estimate of length 
is consistent with binding to sites on separate subunits in the wider, extracellular portion of 
the channel. Nonetheless, it may be the case that in addition to tethering the piperidinyl 
groups, the flexible linker region of the bis-TMP-n compounds serves to stabilize binding 
via hydrophobic interactions, and differences in this form of interaction could underlie 
some of the sensitivity differences between the chimeras and the neuronal receptors. 

An amphipathic analogue of bis-TMP- 10, ATMP-10 was synthesized to evaluate this 
hypothesis. This inhibitor consists of a single piperidinol ring coupled to a 10 carbon 
aliphatic chain and thus contains both a polar head group and nonpolar tail effectively 
making it amphipathic. Interestingly, inhibition by this compound has some striking 
differences when compared with inhibition by bis-TMP- 10. The respective IC50S for long- 
term inhibition of oc3|34 receptors (22 |iM) and alpl(P4TM2)y8 receptors ( 1 uM) by ATMP- 
10 are orders of magnitude higher than the IC50 for inhibition of these receptor subtypes by 
bis-TMP- 10 (200 nM and 30 nM respectively). In addition, prolonged application of high 
concentrations of ATMP-10 in the absence of agonist results in long-term inhibition. It is 
unclear whether sensitivity to this form of inhibition is regulated by the same sequence 
elements which regulate sensitivity to use-dependent inhibition. However, the fact that a 
clear pattern of recovery from this form of inhibition is not observed within the time frame 
of our experiments would seem to argue for interactions of a different nature than those 
observed for inhibition by bis-TMP- 10. In the case of a3(34 receptors, inhibition continues 



138 

to accumulate over time suggesting that the aliphatic chain mediates a hydrophobic 
interaction between the receptor and the inhibitor which does not require the presence of 
agonist. Alternatively, the inhibitor may partition into the membrane and inhibit the 
receptors in a use-dependent manner at later time points upon application of ACh alone; 
however, no noticeable changes in waveform are observed in response to later applications 
of ACh alone making this possibility seem unlikely. The fact that this continued 
accumulation of inhibition is not observed for aipi(p4TM2)y5 receptors suggests that this 
effect may be subtype-dependent. Furthermore, other data from this laboratory indicate 
that wild-type muscle receptors also exhibit agonist-independent inhibition suggesting that 
this form of inhibition does not require the presence of two "sensitive" subunits (R.L. 
Papke, personal communication). 

Additional preliminary results from experiments assessing the effects of intracellularly 
applied ATMP-10 at a concentration of 2 pM indicate that, at this concentration at least, 
there is no detectable inhibition of either a3|34 or al(M((34TM2)y5 receptors (Francis and 
Papke, unpublished observations). Consistent with the amphipathic quality of ATMP-10, 
these data perhaps suggest that a polarity exists for the partitioning of this drug into the 
membrane or, more likely, for binding to hydrophobic sites on the receptor surface itself 
such that the molecule may be unable to pass completely through the oocyte membrane or 
hydrophobic areas of the receptor to gain access to the TMP binding sites which are most 
likely present extracellularly. 

By way of contrast, other experiments in this laboratory indicate that use- or agonist- 
dependent inhibition by ATMP-10 does appear to have a requirement for the presence of 
two "sensitive" subunits. Specifically, neither aip4(piTM2)y8 receptors nor aipi(p4TM2)y 
receptors (lacking the delta subunit) exhibit long-term inhibition after a 10 s co-application 
of 2 uM ATMP-10 with ACh (Francis and Papke, unpublished observations). Therefore, 
it may be the case that prolonged ( 1 minute) drug application in the absence of agonist 
allows the amphipathic inhibitor, in a time-dependent manner, to gain access to sites which 



139 

are unavailable to bis-TMP-10 in the closed configuration of the receptor and thereby 
obviates the requirement for the presence of two "sensitive" subunits. In contrast, 
inhibition arising from short-term application of ATMP-10 in the presence of agonist still 
exhibits this requirement. 

Inhibition by ATMP-10 in the presence of agonist is also demonstrated to be voltage- 
dependent and preapplication of TMP protects aipi((J4TM2)Y5 receptors from long-term 
inhibition by ATMP- 10. As inhibition by TMP has also been shown to mildly voltage- 
dependent, these results may indicate that ATMP-10 and TMP interact with sites deeper in 
the pore than those with which bis-TMP-10 interacts. 

Studies of long chain ammonium compounds including a long chain piperidine 
compound in which the aliphatic chain is connected via the nitrogen of the piperidine ring 
have shown that inhibition of ganglionic receptors by these compounds is voltage- 
dependent as well (Kurenny et al, 1994). In addition, the time course of inhibition by 
these compounds was shown to be dependent on chain length. In this laboratory, 
preliminary studies of ATMP-10 analogues with chain lengths of 4 (ATMP-4) and 7 
carbons (ATMP-7) indicate a similar effect of chain length on inhibition (Francis and 
Papke, unpublished observations). Furthermore, preliminary studies of a compound 
containing a piperidine ring linked to a cyclohexyl ring (the basic nitrogen of the piperidine 
is substituted with a carbon) by a ten carbon aliphatic chain indicate that this compound 
exhibits little inhibition of aipi((34TM2)y5 receptors (Francis and Papke, unpublished 
observations). Therefore, it seems to be the case that two modes of inhibition by the TMP 
compounds exist. Both forms of inhibition are dependent upon the length of the aliphatic 
chain but one form is voltage-dependent while the other is voltage-independent. In the case 
of long chain monofunctional compounds, this chain length requirement most likely reflects 
hydrophobic interactions between the receptor and the aliphatic chain itself whereas in the 
case of the bi-functional compounds, this chain length requirement more likely reflects the 



140 

distance between TMP binding sites in the pentameric receptor although a role for the 
aliphatic linker in contributing to the time course of inhibition cannot be ruled out. 

If the primary function of the linker region is to serve as a tether between active 
inhibitory moieties, then the differences in size constraints may provide some insight into 
the disposition of sensitive subunits within a receptor complex. While a number of studies 
have provided evidence that the delta subunit is situated adjacent to the beta subunit in 
Torpedo nAChRs (Holtzman et al., 1981; Karlin et al, 1983; Machold et al., 1995), 
comparable data is lacking for muscle receptors, either in vivo or in heterologous 
expression systems. Our results demonstrate that both a minimal 10 carbon distance 
between TMP moieties and the presence of the delta subunit are required for effective long- 
term inhibition of aipi((34TM2)y8 receptors. In contrast to the less stringent length 
requirement for long term inhibition of neuronal receptors, the requirement for a long linker 
to achieve long-term inhibition of aipi(P4TM2)yS receptors seems most consistent with a 
model in which the distance between the TMP moieties of an extended conformation of bis- 
TMP-10 represents the distance between opposing rather than adjacent beta and delta 
subunits. Data from studies of rigid analogues of the bis-TMP-n compounds are also 
consistent with this hypothesis (Francis and Papke, unpublished observations). Although 
the lack of voltage-dependence for inhibition of either ap5 or aipi((J4TM2)Y6 receptors 
implies that each putative TMP binding site is located outside of the membrane electric field 
and thus most likely on the extracellular face of the receptor, our data to this point cannot 
absolutely rule out the alternative possibility that the inhibitor binds to asymmetrically 
disposed sites on adjacent subunits in aipi(P4TM2)y5 receptors. In contrast, for ap5 
receptors it is presumed that the formation of a functional receptor requires the presence of 
two agonist binding sites at the interfaces of two sets of alpha-delta pairs. With this 
requirement in mind, the sensitive delta subunits are almost certainly situated in a non- 
adjacent configuration in this receptor subtype. Thus, for the aps subunit combination, it 
seems that bis-TMP-10 is almost certainly binding to non-adjacent subunits. 



141 

In summary, these results demonstrate that the selective long-term inhibition of neuronal 
nicotinic receptors by bis-TMP-10 is dependent upon sequence in the TM2 region. 
However, long-term inhibition apparently occurs independent of binding to this region 
directly. Because inhibition depends upon prior activation of the channel, our results imply 
that bis-TMP-10 binds to a structural element which may become available as a result of the 
conformational change associated with channel gating. The observed compound length 
requirements are consistent with binding to an extracellular site. This form of inhibition by 
bis-TMP-10 contrasts with the form of inhibition observed for QX-3 14 and with 
characteristics reported for inhibition by mecamylamine. Specifically, while sensitivity to 
inhibition by these two compounds seems to be regulated by the same stretch of sequence 
which regulates sensitivity to inhibition by bis-TMP-10, inhibition by both mecamylamine 
and QX-3 14 is profoundly voltage-dependent. Thus, it may be the case that this region of 
TM2 regulates sensitivity to use-dependent inhibitors via two distinct mechanisms: direct 
participation in a binding site for open-channel blockers as would be the case for 
mecamylamine and QX-3 14 and a more indirect exposure of a secondary binding site for 
the binding of voltage-independent inhibitors such as bis-TMP-10. Future studies 
employing this class of inhibitors and functional analogues in conjunction with other 
techniques may add unique insights to the structural changes that take place with channel 
gating. Furthermore, a better understanding of the mechanism of inhibition and structural 
elements which regulate sensitivity to different forms of inhibition may prove valuable in 
the development of agents which act as more potent agonists and antagonists with 
specificity for individual subtypes of neuronal nAChR. 



CHAPTER 5 
SUMMARY AND CONCLUSIONS 



Ion channel function underlies all rapid communication between cells of the nervous 
system. For this reason, the study of ion channels is fundamental to answering some of 
the major questions in the field of neuroscience. In principle, our ability to interact with 
our environment and the nature of this interaction must be encoded at some level by the 
activity of ion channels. In addition to, or perhaps as a result of, their critical role in the 
physiology of the nervous system, ion channels are attractive targets for pharmacological 
manipulation of nervous system function. Nicotinic acetylcholine receptors constitute a 
class of ligand-gated ion channels which are present both in the central and peripheral 
nervous systems as well as at the neuromuscular junction. The studies in this dissertation 
represent an attempt to characterize the relationship between structure and function for 
nicotinic acetylcholine receptors with an emphasis on understanding the changes that take 
place upon receptor activation which result in defining a subtype-selective sensitivity to the 
use-dependent inhibitor bis-TMP-10. Although this drug in and of itself has little clinical 
utility, it is hypothesized that an understanding of the structural basis for inhibition by this 
compound may yield insights which can be applied to the design and characterization of 
more clinically relevant compounds. 

Our results do not indicate an appreciable voltage -dependence for the onset of inhibition 
by bis-TMP-10 (for ap5 and ocipi(P4TM2)y8 receptors in particular) while a profound 
voltage-dependence has been described for inhibition of nAChRs by QX-222 and QX-3 14 
as well as for inhibition by the ganglionic blockers hexamethonium, chlorisondamine and 
mecamylamine. In addition, preapplication of QX-3 14 does not appear to have a great 
effect on inhibition by bis-TMP-10 (for alpl(P4TM2)v5 receptors particularly). As 

142 



143 

inhibition by a number of other use-dependent inhibitors (e.g., QX-222, QX-314, 
mecamylamine) has been demonstrated to arise from a direct, voltage-dependent interaction 
of the inhibitor with this region of TM2, the observations for bis-TMP-10 are somewhat 
novel. From these studies, it appears that the selectivity of bis-TMP-10 arises through the 
exposure of sequence elements outside the N-terminal portion of TM2; however it appears 
that exposure of the site for bis-TMP-10 binding is regulated by sequence within the N- 
terminal portion of TM2. Although our results cannot absolutely rule out a high affinity 
voltage-independent interaction of bis-TMP-10 with elements of TM2 not contained within 
the binding site for QX-314, this possibility seems improbable. Voltage-independent bis- 
TMP-10 effects on the delta subunit seem to be mediated by sequence elements as deep as 
the 6' site which has previously been shown to experience about 78% of the membrane 
electric field. Furthermore, beta subunit-mediated effects of bis-TMP-10 are regulated by 
sequence contained within a region extending from position 1 1' to possibly as deep as 
position 4'. The QX-314 binding site has been suggested to extend from position 6' to 
position 10' (Pascual and Karlin, 1997). Therefore, effects mediated by a direct interaction 
of bis-TMP-10 with the chimeric region would be expected to decrease in the presence of 
high concentrations of QX-3 14. 

If bis-TMP-10 does in fact bind to sequence elements spatially distinct from the region 
of TM2 exchanged in our chimeras, the results imply that drug selectivity may be regulated 
at two different levels: the amino acid composition of the site with which the inhibitor 
interacts and the amino acids which contribute to regulation of exposure of this site. 
Because the interaction between bis-TMP-10 and the region of TM2 exchanged in this 
study appear to be indirect, any hypotheses about the location of the bis-TMP-10 binding 
site from the data in this study are necessarily speculative. Nonetheless, if the 
generalizability of these results to other drugs is to be evaluated, it will be necessary to first 
characterize the sequence elements underlying each of the putative sites of regulation of 
sensitivity to long-term inhibition. For example, it might be predicted that the amino acid 



144 

composition of the bis-TMP-10 binding site itself would primarily affect affinity for the 
inhibitor and therefore time-course of recovery from long-term inhibition, while the amino 
acid composition of the TM2 sequence elements regulating exposure of the bis-TMP-10 
binding sites may affect the quality of inhibition. Specifically, if the sequence elements 
present within the TM2 regions of at least two subunits within a pentameric receptor permit 
exposure of TMP binding sites, this receptor subtype would be predicted to be sensitive to 
long-term inhibition while the exposure of a TMP binding site on only a single subunit 
would be predicted to produce sensitivity to only short-term inhibition. 

Towards describing sequence elements within TM2 which underlie regulation of 
sensitivity, it will be necessary to define single amino acids within the region of the 
neuronal beta subunit chimeras which influence the sensitivity to long-term inhibition by 
bis-TMP-10. From the data presented here, it appears that both the 6' and 10' residues 
may be involved in regulation of the exposure of a site for the binding of bis-TMP-10. In 
fact, more recent data from this laboratory indicate that double mutants exchanging only the 
6' and 10' residues completely recapitulate the effects observed for the beta subunit TM2 
chimeras (R.L. Papke, personal communication). It is interesting to note that these 
residues are also believed to be at sites involved in a direct, voltage-dependent interaction of 
QX-222 and presumably QX-3 14 with the receptor. Thus, it may be the case that, upon 
channel activation, these residues undergo a change in orientation so that they become more 
accessible in the channel pore and, in the process of this reorientation, also alter the 
exposure of sequence elements downstream (extracellular) of TM2. It has been 
demonstrated that the 9' residue is both important in receptor gating and mutation of this 
residue decreases inhibition by QX-222 so a dual role of this type for sequence in this 
region is not unlikely (Filatov and White, 1995; Kearney et ai, 1996; Labarca et ai, 
1995). Other data in support of this hypothesis come from the laboratory of Arthur Karlin. 
Using SCAM analysis, Karlin and co-workers have shown that the pattern of accessibility 
of amino acids contributing to the pore region changes with channel activation and, 



145 

furthermore, that this pattern of accessibility differs between the agonist-binding muscle 
alpha 1 subunit and the structural subunit betal which does not participate in agonist 
binding in muscle nAChRs (Akabas et al; 1994; Zhang and Karlin, 1996). Therefore, it 
may be the case that only subunits which participate as accessory subunits in agonist 
binding undergo the conformational changes associated with exposure of a bis-TMP-10 
binding site whereas structural subunits such as muscle betal do not exhibit this effect. 

It is interesting to contrast this situation with that observed for the delta and gamma 
subunit exchanges. For both of these subunits, the 10' site in the wild-type subunit is a 
nonpolar alanine. Thus, by parallel with the beta subunit effects, it might be expected that 
exchange of the 6' site alone would be completely sufficient to mediate all effects on the 
time-course of recovery from inhibition. However, a(3Y(5-65) mutant receptors have a 
faster time -course of recovery from inhibition than a(i5 receptors, again consistent with a 
model in which, for long-term inhibition, the off-rate of the inhibitor and thus, the time- 
course of return of responsiveness, may depend on sequence at the inhibitor binding site 
itself rather than sequence within TM2. 

From the relatively straightforward characterization of the structural basis for the 
selectivity of bis-TMP-10, we have been able to gain insights into a potential role for the N- 
terminal portion of TM2 in transmitting gating movements. Although the experiments to 
date have not been successful in localizing the bis-TMP-10 binding site per se, they have 
isolated sequence elements which may function to regulate exposure of this site. Whether 
or not this form of inhibition will prove to be generalizable to other pure inhibitors or other 
mixed agonist/antagonists remains to be demonstrated. However, it is interesting to note 
that recent data from this laboratory emphasize the importance of portions of the same 
sequence in the TM2 region of beta subunits in regulation of sensitivity to secondary 
inhibition by nicotine (Webster et ai, unpublished observations). Moreover, the process 
underlying this inhibition appears to be only weakly voltage-dependent. It may be the case 
that the secondary inhibition of certain subtypes nAChRs observed after application of 



146 

nicotine-like experimental therapeutic compounds is also associated with this region of 
TM2 via either a direct, binding interaction or in a more indirect gating-dependent manner. 
In either case, a better understanding of the mechanism of drug action and the sequence 
involved in mediating gating transitions should result from continued characterization of 
this class of noncompetitive inhibitors. This information will be useful in the continuing 
development of specific therapeutic strategies targeting nicotinic receptors. 












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BIOGRAPHICAL SKETCH 

Michael Marvin Francis was born on August 14, 1970, in West Palm Beach, Florida, to 
Sandy and Miles Francis. Soon after Michael's birth, the Francis family moved to 
Jacksonville, Florida where Michael grew up. Michael attended high school at Jacksonville 
Episcopal High School. For his college education, Michael attended the University of 
Virginia where he majored in psychology with a specialization in psychobiology. After a 
year of post-graduate travels, Michael decided to use his brain for more than late night 
philosophy discussions and applied to attend graduate school in the field of neuroscience. 
This decision brought Michael to the University of Florida, where, after a wrong turn or 
two, Michael found himself in the laboratory of Dr. Roger L. Papke studying the function 
of nicotinic acetylcholine receptors. After completing his Ph.D., Michael will continue his 
work in the field of ligand-gated ion channels in the laboratory of Dr. Robert Oswald at 
Cornell University in Ithaca, NY. 



161 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the Degree of Doctor of Philosophy. 





7" ~7<s 
Roger fcTapke, Chair 

Associate Professor of Pharamocology 

and Therapeutics and Neuroscience 



I certify that I have read this study and that in my opinion it conforms to acceptable 



standards of scholarly presentation and is fully adequate, i 
dissertation for the Degree of Doctor of PhilosophyP 



opa and quality, as a 




Peter A.V. Anderson 
Professor of Neuroscience 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the Degree of Doctor of Philosoph) 




c 



\ 




K. Harrison 

nt Professor of Pharmacology and 

peuitcs 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the Degree of Doctor of Philosophy. 










Benjamin A. Horenstein 
Assistant Professor of Chemistry 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the Degree of Doctor of Philosophy. 




du*£(Ci. 



Michael A. King 

Assistant Scientist of Neuroscierice 




This dissertation was submitted to the Graduate Faculty of the College 
of Medicine and to the Graduate School and was accepted a^partiai fulfillment of 
the requirements for the degree of Doctor of Philosoj 

December 1998 







M. Jack Ohanian 

Interim Dean, Graduate School 















UNIVERSITY OF FLORIDA 

3 1262 08555 3146